Methods and compositions for treatment of a beta thalessemia

ABSTRACT

Methods and compositions for treatment of a beta thalessemia are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. ProvisionalApplication No. 61/935,219, filed Feb. 3, 2014, the disclosure of whichis hereby incorporated by reference in its entirety.

TECHNICAL FIELD

The present disclosure is in the field of genome engineering ofhematopoietic stem cells, especially for the treatment of ahemoglobinopathy such as beta thalessemia.

BACKGROUND

Gene therapy holds enormous potential for a new era in human medicine.These methodologies will allow treatment for conditions that heretoforehave not been addressable by standard medical practice. One area that isespecially promising is the ability to genetically engineer a cell tocause that cell to express a product not previously being produced inthat cell, for example due to a mutation that inactivates the cognategene in its genome. Examples of uses of this technology include thetargeted correction of a disease-causing mutation, insertion of a geneencoding a novel therapeutic protein, insertion of a coding sequenceencoding a protein that is lacking in the cell or in the individual,insertion of a wild type gene in a cell containing a mutated genesequence, and insertion of a sequence that encodes a structural nucleicacid such as a microRNA or siRNA.

Transgenes can be delivered to a cell by a variety of ways, such thatthe transgene becomes integrated into the cell's own genome and ismaintained there. In recent years, a strategy for transgene integrationhas been developed that uses cleavage with site-specific nucleases fortargeted insertion into a chosen genomic locus (see, e.g., co-owned U.S.Pat. No. 7,888,121). Nucleases specific for targeted genes can beutilized such that the transgene construct is inserted by eitherhomology directed repair (HDR) or by end capture during non-homologousend joining (NHEJ) driven processes. Targeted loci include “safe harbor”loci for example a CCR5 gene, a CXCR4 gene, a PPP1R12C (also known asAAVS1) gene, an albumin gene or a Rosa gene. See, e.g., U.S. Pat. Nos.7,888,121; 7,972,854; 7,914,796; 7,951,925; 8,110,379; 8,409,861;8,586,526; U.S. Patent Publications 20030232410; 20050208489;20050026157; 20060063231; 20080159996; 201000218264; 20120017290;20110265198; 20130137104; 20130122591; 20130177983 and 20130177960).Nuclease-mediated integration offers the prospect of improved transgeneexpression, increased safety and expressional durability, as compared toclassic integration approaches that rely on random integration of thetransgene, since it allows exact transgene positioning for a minimalrisk of gene silencing or activation of nearby oncogenes. Nucleasesinclude zinc finger nucleases (ZFN), transcription activator likeeffector nucleases (TALENs), mega or homing endonucleases, nucleasesystems such as CRISPR/Cas that use a guide RNA to determinespecificity, and fusions between nucleases such as mega-TALs.

Nuclease-mediated targeted integration can also be used for targetedgene correction of an endogenous locus. Gene correction can occurfollowing nuclease cleavage as above by inserting a transgene ofinterest into the mutant endogenous locus. Alternatively, a mutant genecan be corrected by integrating a portion of the wild type or engineeredsequence to replace (correct) the mutant portion of the gene locus. Forgene correction, as with targeted integration of any exogenous sequence,sequence specific nucleases are used (e.g., ZFN, TALENs, CRISPR/Cas,meganucleases or megaTALs) to introduce a DSB in the endogenous gene ofinterest and a donor, typically comprising homology arms to theendogenous (mutant) gene, is integrated such that the endogenous(mutant) gene is altered by the donor. This approach can be used tocorrect mutations within an endogenous gene sequence and/or to insertengineered sequences for a desired purpose.

Red blood cells (RBCs), or erythrocytes, are the major cellularcomponent of blood. In fact, RBCs account for one quarter of the cellsin a human. Mature RBCs lack a nucleus and many other organelles inhumans, and are full of hemoglobin, a metalloprotein found in RBCs thatfunctions to carry oxygen to the tissues as well as carry carbon dioxideout of the tissues and back to the lungs for removal. The protein makesup approximately 97% of the dry weight of RBCs and it increases theoxygen carrying ability of blood by about seventy fold. Hemoglobin is aheterotetramer comprising two α-like globin chains and two β-like globinchains and 4 heme groups. In adults the α2β2 tetramer is referred to asHemoglobin A (HbA) or adult hemoglobin. Typically, the alpha and betaglobin chains are synthesized in an approximate 1:1 ratio and this ratioseems to be critical in terms of hemoglobin and RBC stabilization. Infact, in some cases where one type of globin gene is inadequatelyexpressed (see below), reducing expression (e.g. using a specific siRNA)of the other type of globin, restoring this 1:1 ratio, alleviates someaspects of the mutant cellular phenotype (see Voon et at (2008)Haematologica 93(8):1288). In a developing fetus, a different form ofhemoglobin, fetal hemoglobin (HbF) is produced which has a higherbinding affinity for oxygen than Hemoglobin A such that oxygen can bedelivered to the baby's system via the mother's blood stream. Fetalhemoglobin also contains two α globin chains, but in place of the adultβ-globin chains, it has two fetal γ-globin chains (i.e., fetalhemoglobin is α2γ2). At approximately 30 weeks of gestation, thesynthesis of γ globin in the fetus starts to drop while the productionof β globin increases. By approximately 10 months of age after birth,the newborn's hemoglobin is nearly all α2β2 although some HbF persistsinto adulthood (approximately 1-3% of total hemoglobin). The regulationof the switch from production of γ to β is quite complex, and primarilyinvolves an expressional down-regulation of γ globin with a simultaneousup-regulation of β globin expression.

Genetic defects in the sequences encoding the hemoglobin chains can beresponsible for a number of diseases known as hemoglobinopathies,including sickle cell anemia and thalassemias. In the majority ofpatients with hemoglobinopathies, the genes encoding γ globin remainintact, but γ globin expression is relatively low due to normal generepression occurring around parturition as described above.

Thalassemias are also diseases relating to hemoglobin and typicallyinvolve a reduced production of globin chains. This can occur throughmutations in the regulatory regions of the genes or from a mutation in aglobin coding sequence that results in reduced production. Alphathalassemias are associated with people of Western Africa and SouthAsian descent, and may confer malarial resistance. Beta thalassemia isassociated with people of Mediterranean descent, typically from Greeceand the coastal areas of Turkey and Italy. Treatment of thalassemiasusually involves blood transfusions and iron chelation therapy. Bonemarrow transplants are also being used for treatment of people withsevere thalassemias if an appropriate donor can be identified, but thisprocedure can have significant risks. Beta thalassemias are dividedgenerally into three groups: i) Thalassemia trait or minor, where thepatients are either carriers of a thalassemia disease allele or havevery mild symptoms that may result in a mild anemia. ii) Thalassemiaintermedia patients, where the lack of beta globin is great enough tocause a moderately severe anemia and significant health problems,including bone deformities and enlargement of the spleen. However, thereis a wide range in the clinical severity of this condition, and theborderline between thalassemia intermedia and the most severe form,thalassemia major, can be confusing. The deciding factor seems to be theamount of blood transfusions required by the patient. iii) ThalassemiaMajor or Cooley's Anemia. This is the most severe form of betathalassemia in which the complete lack of beta globin causes alife-threatening anemia that requires regular blood transfusions andextensive ongoing medical care. These extensive, lifelong bloodtransfusions lead to iron-overload which must be treated with chelationtherapy to prevent early death from organ failure.

For beta thalessemias, in the late 1980s, it was estimated thatapproximately 54 mutations in the beta globin gene encompassed all knowndiseased beta globin genes; the number has since grown to over 200. Themutations are broken down into mutations which result in non-functionalbeta globin protein, including nonsense mutations and frameshiftmutations; RNA processing mutations, including changes in the splicejunctions and changes in splice consensus sequences as well as changesin internal intronic and exonic sequences that result in aberrantsplicing; transcriptional mutants; polyA and RNA cleavage mutants; capsite mutants; and unstable mRNA mutants. These mutations result ineither complete loss of beta globin mRNA in the cell (also referred toas “β−0”) or a reduced level of expression and mRNA accumulation(referred to as “β+”). (see, e.g., Kazazian and Boehm (1988) Blood vol71 No 4: 1107). Patients with the milder β+ forms of beta thalessemiamay have relatively normal lifespans, but those with the severe β−0forms may die before age 30 if untreated, and if iron levels are notmanaged may also have a similarly shortened life span if given frequenttransfusions. Of note, some specific mutations are more common thanothers; in particular, in some parts of Europe and the Middle East, amutation known as “IVS1-1” (which stands for “intervening sequence 1,mutation number 1”) accounts for approximately 20% of all b-thalassemiamajor mutations. In this mutation, the “GT” dinucleotide at thebeginning of intron 1 of the human beta-globin gene is changed to an“AT,” thereby disrupting a key signal (known as the “splice donormotif”) essential for accurate and efficient removal of the intron 1from the beta-globin pre-mRNA. As a result, a significant reduction inbeta-globin protein is observed during erythropoiesis, resulting intransfusion-dependent b-thalassemia major.

Thus, there remains a need for additional methods and compositions thatcan be used for genome editing, to correct an aberrant gene or alter theexpression of others for example to treat beta thalassemias.

SUMMARY

Disclosed herein are methods and compositions for altering theexpression of and/or for correcting one or more genes encoding proteinsinvolved in a genetic disease (e.g., producing proteins lacking,deficient or aberrant in the disease and/or proteins that regulate theseproteins) such as hemoglobinopathies (e.g., beta thalassemias).Alteration of such genes can result in the treatment of these geneticdiseases (e.g., beta thalassemias). In particular, genome editing isused to correct an aberrant gene, insert a wild type gene, or change theexpression of an endogenous gene. By way of non-limiting example, amutated gene encoding β globin may be corrected in a cell to produce awild type β globin protein in the cell to treat a hemoglobinopathycaused by the faulty β globin gene. One approach further involves theuse of modification of a stem cell (e.g., hematopoietic stem cell or RBCprecursor), which stem cell can then be used to engraft into a patient,for treatment of a hemoglobinopathy.

In one aspect, described herein is a nuclease protein or system (e.g.,ZFN, TALEN, mega or homing endonuclease, mega-TAL or a CRISPR/Cassystem) that binds to target site in a region of interest (e.g., a βglobin gene) in a genome, wherein the nuclease comprises one or moreengineered domains. In one embodiment, the ZFP is a zinc-finger nuclease(ZFN) that cleaves a target genomic region of interest, wherein the ZFNcomprises one or more engineered zinc-finger binding domains and anuclease cleavage domain or cleavage half-domain. In another embodiment,the nuclease is a TALE nuclease (TALEN) that cleaves a target genomicregion of interest, wherein the TALEN comprises one or more engineeredTALE DNA binding domains and a nuclease cleavage domain or cleavagehalf-domain. In another embodiment, the nuclease is a CRISPR/Cas systemwherein the specificity of the CRISPR/Cas is determined by an engineeredsingle guide mRNA. Cleavage domains and cleavage half domains can beobtained, for example, from various restriction endonucleases and/orhoming endonucleases. In one embodiment, the cleavage half-domains arederived from a Type IIS restriction endonuclease (e.g., Fok I). Incertain embodiments, the DNA binding domain (e.g. zinc finger or TALEDNA binding domain) recognizes a target site in a beta globin gene. Incertain embodiments, the zinc finger domain comprises 5 or 6 zinc fingerdomains and recognizes a target site in a globin gene.

In another aspect, described herein is a CRISPR/Cas system that binds totarget site in a region of interest (e.g., a highly expressed gene, adisease associated gene or a safe harbor gene) in a genome, wherein theCRISPR/Cas system comprises a CRIPSR/Cas nuclease and an engineeredcrRNA/tracrRNA (or single guide RNA). In certain embodiments, theCRISPR/Cas system recognizes a target site in a highly expressed,disease associated, or safe harbor gene. In certain embodiments, theCRISPR/Cas system recognizes a target in a beta globin gene.

The ZFNs, TALENs and/or CRISPR/Cas system as described herein may bindto and/or cleave the region of interest in a coding or non-coding regionwithin or adjacent to the gene, such as, for example, a leader sequence,trailer sequence or intron, or within a non-transcribed region, eitherupstream or downstream of the coding region. In certain embodiments, theZFNs, TALENs and/or CRISPR/Cas system bind(s) to and/or cleave(s) aglobin gene. In other embodiments, the ZFNs, TALENs and/or CRISPR/Cassystem binds to and/or cleaves a safe-harbor gene, for example a CCR5gene, a CXCR4 gene, a PPP1R12C (also known as AAVS1) gene, an albumingene, an HPRT gene or a Rosa gene. See, e.g., U.S. Pat. Nos. 7,888,121;7,972,854; 7,914,796; 7,951,925; 8,110,379; 8,409,861; 8,586,526; U.S.Patent Publications 20030232410; 20050208489; 20050026157; 20060063231;20080159996; 201000218264; 20120017290; 20110265198; 20130137104;20130122591; 20130177983 and 20130177960. In addition, to aid inselection, the HPRT locus may be used (see U.S. Patent Publication No.20130122591). In another aspect, described herein are compositionscomprising one or more of the zinc-finger and/or TALE nucleases orCRISPR/Cas system as described herein. In some embodiments, the ZFNs,TALENs and/or CRISPR/Cas system binds to and cleaves a β or globin gene.In another aspect, described herein are compositions comprising one ormore of the zinc-finger, TALE or Cas nucleases as described herein.

In another aspect, described herein is a polynucleotide encoding one ormore ZFNs, TALENs and/or CRISPR/Cas system as described herein. Thepolynucleotide may be, for example, mRNA. In some aspects, the mRNA maybe chemically modified (See e.g. Kormann et al, (2011) NatureBiotechnology 29(2):154-157). In other aspects, the mRNA may comprise anARCA cap (see U.S. Pat. Nos. 7,074,596 and 8,153,773). In furtherembodiments, the mRNA may comprise a mixture of unmodified and modifiednucleotides (see U.S. Patent Publication 2012-0195936).

In another aspect, described herein is a ZFN, TALEN and/or CRISPR/Cassystem expression vector comprising a polynucleotide, encoding one ormore ZFNs, TALENs and/or CRISPR/Cas system described herein, operablylinked to a promoter. In one embodiment, the expression vector is aviral vector.

In one aspect, described herein is a ZFN, TALEN and/or CRISPR/Cas systemprotein that is used to cleave a target DNA.

In another aspect, described herein is a genetically modified cell orcell line, for example as compared to the wild-type sequence of the cellor cell line. In certain embodiments, the cell comprises geneticallymodified RBC precursors (hematopoietic stem cells known as “HSCs”). Thecell or cell lines may be heterozygous or homozygous for themodification. The modifications may comprise insertions, deletionsand/or combinations thereof. In certain embodiments, the HSCs aremodified with an engineered nuclease and a donor nucleic acid such thata wild type gene (e.g., globin gene) is inserted and expressed and/or anendogenous aberrant gene is corrected. In certain embodiments, themodification (e.g., insertion) is at or near the nuclease(s) bindingand/or cleavage site(s), for example, within 1-300 (or any valuetherebetween) base pairs upstream or downstream of the site(s) ofcleavage, more preferably within 1-100 base pairs (or any valuetherebetween) of either side of the binding and/or cleavage site(s),even more preferably within 1 to 50 base pairs (or any valuetherebetween) on either side of the binding and/or cleavage site(s). Insome cases, the wild type gene sequence for insertion encodes a wildtype 0 globin. In other cases, the endogenous aberrant gene is the βglobin gene, for example one or more genomic modifications that correctat least one mutation in an endogenous aberrant human beta-hemoglobin(Hbb) gene. In some aspects, the modification of the beta globin geneallows proper splicing of transcribed RNAs. In some cases, the regionfor correction comprises the IVS 1-1, 1-5, 1-6, 1-110 mutations. In apreferred embodiment, the beta globin region that is corrected comprisesthe IVS 1-1 mutation. In some embodiments, the region for correctioncomprises IVS 2-1, 2-745, or 2-654 mutations. Partially or fullydifferentiated cells descended from the modified stem cells as describedherein are also provided (e.g., RBCs or RBC precursor cells).Compositions such as pharmaceutical compositions comprising thegenetically modified cells as described herein are also provided.

In another aspect, described herein is a method for correcting orinserting a sequence into an endogenous gene (e.g., a beta globin gene)in a cell (e.g., stem cell), the method comprising cleaving theendogenous gene using one or more nucleases and correcting or insertinga sequence into the cleavage site. In certain embodiments, a genomicsequence in any target gene is replaced, for example using a ZFN orTALEN pair, or a CRIPSR/Cas system (or vector encoding said ZFN, TALENand/or CRIPSR/Cas system) as described herein and a “donor” sequence (insome embodiments also known as a “transgene”) that is inserted into thegene following targeted cleavage with the ZFN, TALEN and/or a CRIPSR/Cassystem. The donor sequence may be present in the ZFN or TALEN vector,present in a separate vector (e.g., Ad, AAV or LV vector) or,alternatively, may be introduced into the cell using a different nucleicacid delivery mechanism. Such insertion of a donor nucleotide sequenceinto the target locus (e.g., globin gene, other safe-harbor gene, etc.)results in the expression of the transgene under control of the targetlocus's (e.g. globin's) genetic control elements. In some embodiments,the transgene encodes a non-coding RNA (e.g., an shRNA).

In other aspects, genetically modified RBC precursors (hematopoieticstem cells known as “HSCs”) are given in a bone marrow transplant andthe RBCs differentiate and mature in vivo. In some embodiments, the HSCsare isolated following G-CSF-induced mobilization, and in others, thecells are isolated from human bone marrow or umbilical cords. In someembodiments, the modified HSCs are administered to the patient followingmild myeloablative pre-conditioning. In other aspects, the HSCs areadministered after full myeloablation such that following engraftment,100% of the hematopoietic cells are derived from the modified HSCs. Inyet another aspect, provided herein are cell lines and/or transgenicanimal models (systems). In some embodiments, the transgenic cell and/oranimal includes a transgene that encodes a human gene. In someinstances, the transgenic animal comprises a knock-out at the endogenouslocus corresponding to exogenous transgene (e.g., the mouse globin geneis knocked out and the human globin gene is inserted into a mouse),thereby allowing the development of an in vivo system where the humanprotein may be studied in isolation. Such transgenic models may be usedfor screening purposes to identify small molecules or large biomoleculesor other entities which may interact with or modify the human protein ofinterest. In some aspects, the transgene is integrated into the selectedlocus (e.g., globin or safe-harbor) into a stem cell (e.g., an embryonicstem cell, an induced pluripotent stem cell, a hematopoietic stem cell,etc.) or animal embryo obtained by any of the methods described herein,and then the embryo is implanted such that a live animal is born. Theanimal is then raised to sexual maturity and allowed to produceoffspring wherein at least some of the offspring comprise editedendogenous gene sequence or the integrated transgene.

In a still further aspect, provided herein is a method for site specificintegration of a nucleic acid sequence into an endogenous locus orcorrection of an endogenous gene at a desired locus (e.g., globin gene)of a chromosome, for example into the chromosome of an embryo. Incertain embodiments, the method comprises: (a) injecting an embryo with(i) at least one DNA vector, wherein the DNA vector comprises anupstream sequence and a downstream sequence flanking the nucleic acidsequence to be integrated, and (ii) at least one RNA molecule encoding azinc finger, TALE or Cas9 nuclease. In the case of using a Cas9 protein,an engineered single guide (sg)RNA is also introduced. The nuclease ornuclease system recognizes the target site in the target locus (e.g.,globin or safe harbor locus), and then (b) the embryo is cultured toallow expression of the zinc finger or TALE nuclease and/or CRISPR/Cassystem, wherein a double stranded break is introduced into the target bythe zinc finger nuclease, TALEN or CRISPR/Cas system is then repaired,via homologous recombination with the DNA vector, so as to integrate thenucleic acid sequence into the chromosome.

In any of the methods described herein, the polynucleotide encoding thezinc finger nuclease(s), TALEN(s) and/or CRIPSR/Cas system can compriseDNA, RNA or combinations thereof. In certain embodiments, thepolynucleotide comprises a plasmid. In other embodiments, thepolynucleotide encoding the nuclease comprises mRNA.

A kit, comprising the ZFPs, TALENs and/or CRIPSR/Cas system of theinvention, is also provided. The kit may comprise nucleic acids encodingthe ZFPs, TALENs or CRISPR/Cas system, (e.g. RNA molecules or ZFP, TALENor Cas9 encoding genes contained in a suitable expression vector) andengineered sg RNA if needed, or aliquots of the nuclease proteins, donormolecules, suitable host cell lines, instructions for performing themethods of the invention, and the like.

Thus, the disclosure includes, but is not limited to, a geneticallymodified cell comprising a genomic modification. In one embodiment, thegenomic modification corrects at least one mutation in an endogenousaberrant human beta-hemoglobin (Hbb) gene. In a further embodiment, thegenomic modification is selected from the group consisting ofinsertions, deletions and combinations thereof. In a related embodiment,the genetically modified cell is made by a nuclease including one ofmore of the nucleases disclosed herein (e.g., ZFN (e.g., as disclosed inthe last two rows of Table 1), TALEN, etc.), which nuclease may beintroduced in nucleic acid and/or protein form. In certain embodiments,the mutation is in an intervening sequence 1, mutation number 1 (IVS1-1)mutation, an IVS1-5 mutation, an IVS1-6 mutation, an IVS1-110 mutation,an IVS2-1 mutation, an IVS2-745 mutation or an IVS2-654 mutation. Inother embodiments, the genomic modification corrects a point mutation.In still further embodiments, the invention comprises the geneticallymodified cell described above, wherein the genomic modification iswithin one or more of the sequences shown in SEQ ID NO:3, for example, agenomic modification within SEQ ID NO: 3 disrupts a splice donor motif.In still further embodiments, the GT dinucleotide at the beginning ofintron 1 of the human beta-globin gene is changed to an AT. In stillfurther embodiments, the genomic modification corrects a mutation thatalters the splice donor site for mRNA processing of the aberrant Hbbgene that leads to beta-thalassemia. In still further embodiments, thegenomic modification is at or near any of the sequences shown as SEQ IDNos. 4 or 5. In still further embodiments, the genomic modificationcomprises insertion of donor sequence comprising a novel restrictionenzyme cleavage site with respect to the endogenous Hbb gene. In certainembodiments, the donor sequence is between 2 kb to 200 kb in length, forexample, a donor sequence selected from any one of SEQ ID Nos. 20 and 22and the novel restriction enzyme cleavage site is an Xba I site. Incertain embodiments, the donor sequence comprises a transgene of anylength.

Any of the genetically modified cells has described herein may be, forexample, a stem cell such as a hematopoietic stem cell (e.g., CD34+).Furthermore, described herein is a genetically modified differentiatedcell descended from any of the genetically modified stem cells asdescribed herein, including, for example, a red blood cell (RBC).

Also provided is a pharmaceutical composition comprising one of more ofthe genetically modified cells as described herein.

In addition, a zinc finger protein comprising 4, 5, or 6 zinc fingerdomains comprising a recognition helix region, each zinc finger domaincomprising the recognition helix regions in the order shown in the lasttwo rows of Table 1 is also provided. In certain embodiments, a fusionprotein comprising a zinc finger protein and a wild-type or engineeredcleavage half-domain is provided. Polynucleotides encoding any of theZFPs and/or fusion proteins described herein are also provided.Described herein are isolated cells (e.g., red blood cells (RBCs) orprecursor cells (e.g., CD4+ hematopoietic stem cells) comprising one ormore the ZFPs, fusion proteins and/or polynucleotides described hereinare also provided. Kits comprising a polynucleotide, a protein and/orcell as described herein are also provided.

Also described is a method of altering Hbb expression in a cell, themethod comprising: introducing, into the cell, one or morepolynucleotides as described herein, under conditions such that the oneor more proteins are expressed and expression of the Hbb gene is altered(e.g., increased). In certain embodiments, the methods further compriseintegrating a donor sequence into the genome of the cell, for exampleusing a viral vector, as an oligonucleotide and/or on a plasmid. Thedonor sequence may comprise a transgene and expression of the transgenemay be under the control of an endogenous or exogenous promoter. Inother embodiments, the donor comprises an oligonucleotide that correctsa mutation that alters the splice donor site for mRNA processing of theHbb gene. In certain embodiments, the cell is a red blood cell (RBC)precursor cell and/or a hematopoietic stem cell. In certain embodiments,the methods comprise producing a genetically modified cell comprising agenomic modification within an endogenous Hbb gene, the methodcomprising the steps of: a) contacting a cell with a polynucleotideencoding a fusion protein comprising a zinc finger nuclease comprising4, 5, or 6 zinc finger domains, each zinc finger domain comprising arecognition helix region and further wherein the recognition helixregions are in the order shown in one of the last two rows of Table 1;b) subjecting the cell to conditions conducive to expressing the fusionprotein from the polynucleotide; and c) modifying the endogenous Hbbgene with the expressed fusion protein sufficient to produce thegenetically modified cell. In any of the methods described herein, themethods may further comprise stimulating the cells with at least onecytokine. The polynucleotide may delivered inside the cell, for example,using at least one of a non-viral delivery system, a viral deliverysystem, and/or a delivery vehicle. In certain embodiments, the methodsinvolve subjecting the cells to an electric field.

Also described is a method of treating a patient with β-thalassemia, themethod comprising administering to the patient the pharmaceuticalpreparation as described herein (e.g., a pharmaceutical compositiondescribes one or more proteins, polynucleotides and/or cells asdescribed herein) in an amount sufficient to increase the Hbb geneexpression in the patient.

These and other aspects will be readily apparent to the skilled artisanin light of disclosure as a whole.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of the human beta-globin gene structure,adapted from Kazazian and Boehm (1998) ibid. Across the top is theillustration of the beta globin gene where the hatched areas indicatethe exons, and the clear areas depict the introns as well as the 5′ and3′ non-coding regions. Also depicted are several of the known types ofbeta globin mutations associated with thalessemias, where an openballoon marker indicates mutations that effect splicing, closed balloonsindicate transcriptional mutations, open squares indicate the cap sitemutation, downward arrow indicates RNA cleavage mutations, upward filledarrows indicate frameshift mutations, upward open arrows indicatenonsense mutations, a block with an asterisk in it indicates mutationsthat increase mRNA instability, and the open rectangles indicateobserved deletion mutations. The bottom panel of FIG. 1 (SEQ ID NO:1)depicts the location of the IVS 1-1 mutation and the wild type sequencecorresponding to this sequence. Also shown are the binding locations fortwo ZFNs to cleave at or near the IVS 1-1 mutation, and the pointmutation responsible for the IVS 1-1 mutation is indicated by an openbracket.

FIGS. 2A and 2B depict the activity of ZFN pair 43545/43544. FIG. 2A isa gel showing cleavage activity of the ZFN pair in human CD34+ cellsusing the Cel I Surveyor Assay, while FIG. 2B is a graph depictingcleavage at either the wild type (“wt”) or the IVS 1.1 mutant sequenceas measured by the Dual-Luciferase Single Strand Annealing Assay (SeeU.S. Pat. No. 8,586,526). The experiments shown in FIG. 2B areduplicates. Control experiments were done with cells transduced with aGFP reporter plasmid (“GFP”) or sham transfected cells (“negative”). Thedata indicates that both alleles are cleaved by the ZFN pair.

FIGS. 3A and 3B depict schematics for introducing a IVS 1-1 mutationinto a wild type beta globin gene. FIG. 3A depicts the steps of the genemodification where the wild type sequence in the endogenous beta globingene is replaced with the IVS 1-1 mutation. The donor is a singlestranded oligonucleotide comprising the IVS 1-1 mutation (depicting theA point mutation) as well as an XbaI restriction site (“ssoligonucleotide”), and the ZFN cleavage site. The cleaved endogenousgene (depicting the wild type G) is shown below the donor nucleotide,and the “gene corrected” endogenous gene, now comprising both the IVS1-1 mutation and the Xbal restriction site, is shown on the bottom. FIG.3B (SEQ ID NO:2) is a close up of the ss oligonucleotide donor, showingthe sequence around the ZFN cleavage site as well as the bracketedintroduced nucleotides.

FIG. 4 depicts the results of the introduction of the IVS 1-1 mutationdescribed in FIG. 3 into the wild type beta globin gene in normal CD34+cells. Shown is a gel depicting the cleavage results from the introducedXbaI restriction enzyme (RFLP). The enzyme cleavage indicates that theCD34+ cells were “corrected” with either a ss oligonucleotide donorcomprising the wild type (“wt”) or IVS 1-1 beta globin sequence and anXbaI restriction site. Also shown below the gel are the results fromhigh throughput sequencing of PCR products that comprise this region inthe treated cells. Indicated is the amount of targeted integration ofthe donor in samples receiving the donor (“% TI”), and the amount of ZFNactivity (“% indels”) in the samples.

FIG. 5 depicts the genomic sequence (SEQ ID NO:3) around the IVS 1-1mutation (Adapted from Lapoumeroulie et al, (1987) Nucleic AcidsResearch, 15(20): 8195). Indicated in the figure are cryptic splicesites that are used when the normal splice site is disrupted by the G->AIVS1-1 mutation.

FIGS. 6A and 6B depict results following gene correction inpatient-derived CD34+ cells. Beta thalassemia patient (“p13” shown inFIG. 6B)-derived CD34+ cells, along with wild type CD34+ cells (‘WT’shown in FIG. 6A) were treated with an oligonucleotide to correct theIVS 1-1 mutation into wild type. Cells were treated with either pairs ofZFNs alone, with ZFNs plus gene correcting donor oligo (“WT oligo”) orwith donor oligo and GFP alone. In most instances, the donor oligo alsocomprised a unique XbaI restriction site (“XbaI”). The gels depict XbaIcleavage of genomic DNA isolated from the treated cells, and demonstrateinsertion of the donor oligonucleotide. Lane designations are asfollows: Lane 1 shows results using ZFNs 43545/43544; Lane 2 showsresults using ZFNs 43545/45946; Lane 3 shows results using ZFNs43545/45952; Lane 4 shows results using ZFNs 43545/43544 and WT oligoXbaI; Lane 5 shows results using ZFNs 43545/45946 and WT oligo XbaI;Lane 6 shows results using ZFNs 43545/45952 and WT oligo XbaI; Lane 7shows results using GFP and WT oligo XbaI; Lane 8 shows results usingZFNs 43545/43544 and WT Oligo (p13 only); and Lane 9 shows results ofuntransfected cells.

DETAILED DESCRIPTION

Disclosed herein are methods and compositions for studying and treatinga genetic disease such as a hemoglobinopathy. The invention describesgenomic editing of a target cell such that there is a favorable changein the production of one or more globin genes, which in turn results intreatment of hemoglobinopathies such as a thalassemia in a subject inneed thereof. Favorable changes in the production of globin includes,but is not limited, correction of an aberrant β globin gene sequence.Additionally, delivery of altered hematopoietic stem cells in atransplant altered to express a desired protein product can be similarlybeneficial in treating hemoglobinopathies such as a thalassemia. Alsodescribed are cell lines and animals with altered globin production.

Thus, the methods and compositions of the invention can be used to alterthe production of one or more globin genes (e.g. β) in a cell (e.g., anerythroid precursor cell). These alterations and the methods andcompositions can be used to edit an endogenous gene or insert a gene ata desired location in the genome of a cell (e.g., into an HSC).Precursor cells can be derived from subjects in need, modified ex vivo,and then given back to the subject either in a bone marrow graft.

General

Practice of the methods, as well as preparation and use of thecompositions disclosed herein employ, unless otherwise indicated,conventional techniques in molecular biology, biochemistry, chromatinstructure and analysis, computational chemistry, cell culture,recombinant DNA and related fields as are within the skill of the art.These techniques are fully explained in the literature. See, forexample, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Secondedition, Cold Spring Harbor Laboratory Press, 1989 and Third edition,2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley& Sons, New York, 1987 and periodic updates; the series METHODS INENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE ANDFUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS INENZYMOLOGY, Vol. 304, “Chromatin” (P. M. Wassarman and A. P. Wolffe,eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULARBIOLOGY, Vol. 119, “Chromatin Protocols” (P. B. Becker, ed.) HumanaPress, Totowa, 1999.

DEFINITIONS

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areused interchangeably and refer to a deoxyribonucleotide orribonucleotide polymer, in linear or circular conformation, and ineither single- or double-stranded form. For the purposes of the presentdisclosure, these terms are not to be construed as limiting with respectto the length of a polymer. The terms can encompass known analogues ofnatural nucleotides, as well as nucleotides that are modified in thebase, sugar and/or phosphate moieties (e.g., phosphorothioatebackbones). In general, an analogue of a particular nucleotide has thesame base-pairing specificity; i.e., an analogue of A will base-pairwith T.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues. The termalso applies to amino acid polymers in which one or more amino acids arechemical analogues or modified derivatives of correspondingnaturally-occurring amino acids.

“Binding” refers to a sequence-specific, non-covalent interactionbetween macromolecules (e.g., between a protein and a nucleic acid). Notall components of a binding interaction need be sequence-specific (e.g.,contacts with phosphate residues in a DNA backbone), as long as theinteraction as a whole is sequence-specific. Such interactions aregenerally characterized by a dissociation constant (K_(d)) of 10⁻⁶ M⁻¹or lower. “Affinity” refers to the strength of binding: increasedbinding affinity being correlated with a lower K_(d).

A “binding protein” is a protein that is able to bind non-covalently toanother molecule. A binding protein can bind to, for example, a DNAmolecule (a DNA-binding protein), an RNA molecule (an RNA-bindingprotein) and/or a protein molecule (a protein-binding protein). In thecase of a protein-binding protein, it can bind to itself (to formhomodimers, homotrimers, etc.) and/or it can bind to one or moremolecules of a different protein or proteins. A binding protein can havemore than one type of binding activity. For example, zinc fingerproteins have DNA-binding, RNA-binding and protein-binding activity.

A “zinc finger DNA binding protein” (or binding domain) is a protein, ora domain within a larger protein, that binds DNA in a sequence-specificmanner through one or more zinc fingers, which are regions of amino acidsequence within the binding domain whose structure is stabilized throughcoordination of a zinc ion. The term zinc finger DNA binding protein isoften abbreviated as zinc finger protein or ZFP.

A “TALE DNA binding domain” or “TALE” is a polypeptide comprising one ormore TALE repeat domains/units. The repeat domains are involved inbinding of the TALE to its cognate target DNA sequence. A single “repeatunit” (also referred to as a “repeat”) is typically 33-35 amino acids inlength and exhibits at least some sequence homology with other TALErepeat sequences within a naturally occurring TALE protein. See, e.g.,U.S. Patent Publication No. 20110301073.

Zinc finger and TALE binding domains can be “engineered” to bind to apredetermined nucleotide sequence, for example via engineering (alteringone or more amino acids) of the recognition helix region of a naturallyoccurring zinc finger or TALE protein. Therefore, engineered DNA bindingproteins (zinc fingers or TALEs) are proteins that are non-naturallyoccurring. Non-limiting examples of methods for engineering DNA-bindingproteins are design and selection. A designed DNA binding protein is aprotein not occurring in nature whose design/composition resultsprincipally from rational criteria. Rational criteria for design includeapplication of substitution rules and computerized algorithms forprocessing information in a database storing information of existing ZFPand/or TALE designs and binding data. See, for example, U.S. Pat. Nos.6,140,081; 6,453,242; and 6,534,261; see also WO 98/53058; WO 98/53059;WO 98/53060; WO 02/016536 and WO 03/016496 and U.S. Publication No.20110301073.

A “selected” zinc finger protein or TALE is a protein not found innature whose production results primarily from an empirical process suchas phage display, interaction trap or hybrid selection. See e.g., U.S.Pat. Nos. 8,586,526; 5,789,538; 5,925,523; 6,007,988; 6,013,453; and6,200,759.

“TtAgo” is a prokaryotic Argonaute protein thought to be involved ingene silencing. TtAgo is derived from the bacteria Thermus thermophilus.See, e.g., Swarts et al, ibid, G. Sheng et al., (2013) Proc. Natl. Acad.Sci. U.S.A. 111, 652). A “TtAgo system” is all the components requiredincluding, for example, guide DNAs for cleavage by a TtAgo enzyme.

“Recombination” refers to a process of exchange of genetic informationbetween two polynucleotides. For the purposes of this disclosure,“homologous recombination” (HR) refers to the specialized form of suchexchange that takes place, for example, during repair of double-strandbreaks in cells via homology-directed repair mechanisms. This processrequires nucleotide sequence homology, uses a “donor” molecule totemplate repair of a “target” molecule (i.e., the one that experiencedthe double-strand break), and is variously known as “non-crossover geneconversion” or “short tract gene conversion,” because it leads to thetransfer of genetic information from the donor to the target. Withoutwishing to be bound by any particular theory, such transfer can involvemismatch correction of heteroduplex DNA that forms between the brokentarget and the donor, and/or “synthesis-dependent strand annealing,” inwhich the donor is used to re-synthesize genetic information that willbecome part of the target, and/or related processes. Such specialized HRoften results in an alteration of the sequence of the target moleculesuch that part or all of the sequence of the donor polynucleotide isincorporated into the target polynucleotide.

In the methods of the disclosure, one or more targeted nucleases asdescribed herein create a double-stranded break in the target sequence(e.g., cellular chromatin) at a predetermined site, and a “donor”polynucleotide, having homology to the nucleotide sequence in the regionof the break, can be introduced into the cell. The presence of thedouble-stranded break has been shown to facilitate integration of thedonor sequence. The donor sequence may be physically integrated or,alternatively, the donor polynucleotide is used as a template for repairof the break via homologous recombination, resulting in the introductionof all or part of the nucleotide sequence as in the donor into thecellular chromatin. Thus, a first sequence in cellular chromatin can bealtered and, in certain embodiments, can be converted into a sequencepresent in a donor polynucleotide. Thus, the use of the terms “replace”or “replacement” can be understood to represent replacement of onenucleotide sequence by another, (i.e., replacement of a sequence in theinformational sense), and does not necessarily require physical orchemical replacement of one polynucleotide by another.

In any of the methods described herein, additional pairs of zinc-fingeror TALEN proteins can be used for additional double-stranded cleavage ofadditional target sites within the cell. In addition, a CRISPR/Cassystem may be similarly employed to induce additional double strandbreaks.

In certain embodiments of methods for targeted recombination and/orreplacement and/or alteration of a sequence in a region of interest incellular chromatin, a chromosomal sequence is altered by homologousrecombination with an exogenous “donor” nucleotide sequence. Suchhomologous recombination is stimulated by the presence of adouble-stranded break in cellular chromatin, if sequences homologous tothe region of the break are present.

In any of the methods described herein, the exogenous nucleotidesequence (the “donor sequence” or “transgene”) can contain sequencesthat are homologous, but not identical, to genomic sequences in theregion of interest, thereby stimulating homologous recombination toinsert a non-identical sequence in the region of interest. Thus, incertain embodiments, portions of the donor sequence that are homologousto sequences in the region of interest exhibit between about 80 to 99%(or any integer therebetween) sequence identity to the genomic sequencethat is replaced. In other embodiments, the homology between the donorand genomic sequence is higher than 99%, for example if only 1nucleotide differs as between donor and genomic sequences of over 100contiguous base pairs. In certain cases, a non-homologous portion of thedonor sequence can contain sequences not present in the region ofinterest, such that new sequences are introduced into the region ofinterest. In these instances, the non-homologous sequence is generallyflanked by sequences of 50-1,000 base pairs (or any integral valuetherebetween) or any number of base pairs greater than 1,000, that arehomologous or identical to sequences in the region of interest. In otherembodiments, the donor sequence is non-homologous to the first sequence,and is inserted into the genome by non-homologous recombinationmechanisms.

Any of the methods described herein can be used for partial or completeinactivation of one or more target sequences in a cell by targetedintegration of donor sequence that disrupts expression of the gene(s) ofinterest. Cell lines with partially or completely inactivated genes arealso provided.

Furthermore, the methods of targeted integration as described herein canalso be used to integrate one or more exogenous sequences. The exogenousnucleic acid sequence can comprise, for example, one or more genes orcDNA molecules, or any type of coding or non-coding sequence, as well asone or more control elements (e.g., promoters). In addition, theexogenous nucleic acid sequence may produce one or more RNA molecules(e.g., small hairpin RNAs (shRNAs), inhibitory RNAs (RNAis), microRNAs(miRNAs), etc.).

“Cleavage” refers to the breakage of the covalent backbone of a DNAmolecule. Cleavage can be initiated by a variety of methods including,but not limited to, enzymatic or chemical hydrolysis of a phosphodiesterbond. Both single-stranded cleavage and double-stranded cleavage arepossible, and double-stranded cleavage can occur as a result of twodistinct single-stranded cleavage events. DNA cleavage can result in theproduction of either blunt ends or staggered ends. In certainembodiments, fusion polypeptides are used for targeted double-strandedDNA cleavage.

A “cleavage half-domain” is a polypeptide sequence which, in conjunctionwith a second polypeptide (either identical or different) forms acomplex having cleavage activity (preferably double-strand cleavageactivity). The terms “first and second cleavage half-domains;” “+ and −cleavage half-domains” and “right and left cleavage half-domains” areused interchangeably to refer to pairs of cleavage half-domains thatdimerize.

An “engineered cleavage half-domain” is a cleavage half-domain that hasbeen modified so as to form obligate heterodimers with another cleavagehalf-domain (e.g., another engineered cleavage half-domain). See, also,U.S. Pat. Nos. 7,888,121; 7,914,796; 8,034,598 and 8,823,618,incorporated herein by reference in their entireties.

The term “sequence” refers to a nucleotide sequence of any length, whichcan be DNA or RNA; can be linear, circular or branched and can be eithersingle-stranded or double stranded. The term “donor sequence” refers toa nucleotide sequence that is inserted into a genome. A donor sequencecan be of any length, for example between 2 and 10,000 nucleotides inlength (or any integer value therebetween or thereabove), preferablybetween about 100 and 1,000 nucleotides in length (or any integertherebetween), more preferably between about 200 and 500 nucleotides inlength.

A “disease associated gene” is one that is defective in some manner in amonogenic disease. Non-limiting examples of monogenic diseases includesevere combined immunodeficiency, cystic fibrosis, lysosomal storagediseases (e.g. Gaucher's, Hurler's, Hunter's, Fabry's, Neimann-Pick,Tay-Sach's, etc.), sickle cell anemia, and thalassemia.

“Chromatin” is the nucleoprotein structure comprising the cellulargenome. Cellular chromatin comprises nucleic acid, primarily DNA, andprotein, including histones and non-histone chromosomal proteins. Themajority of eukaryotic cellular chromatin exists in the form ofnucleosomes, wherein a nucleosome core comprises approximately 150 basepairs of DNA associated with an octamer comprising two each of histonesH2A, H2B, H3 and H4; and linker DNA (of variable length depending on theorganism) extends between nucleosome cores. A molecule of histone H1 isgenerally associated with the linker DNA. For the purposes of thepresent disclosure, the term “chromatin” is meant to encompass all typesof cellular nucleoprotein, both prokaryotic and eukaryotic. Cellularchromatin includes both chromosomal and episomal chromatin.

A “chromosome,” is a chromatin complex comprising all or a portion ofthe genome of a cell. The genome of a cell is often characterized by itskaryotype, which is the collection of all the chromosomes that comprisethe genome of the cell. The genome of a cell can comprise one or morechromosomes.

An “episome” is a replicating nucleic acid, nucleoprotein complex orother structure comprising a nucleic acid that is not part of thechromosomal karyotype of a cell. Examples of episomes include plasmidsand certain viral genomes.

A “target site” or “target sequence” is a nucleic acid sequence thatdefines a portion of a nucleic acid to which a binding molecule willbind, provided sufficient conditions for binding exist.

An “exogenous” molecule is a molecule that is not normally present in acell, but can be introduced into a cell by one or more genetic,biochemical or other methods. “Normal presence in the cell” isdetermined with respect to the particular developmental stage andenvironmental conditions of the cell. Thus, for example, a molecule thatis present only during embryonic development of muscle is an exogenousmolecule with respect to an adult muscle cell. Similarly, a moleculeinduced by heat shock is an exogenous molecule with respect to anon-heat-shocked cell. An exogenous molecule can comprise, for example,a functioning version of a malfunctioning endogenous molecule or amalfunctioning version of a normally-functioning endogenous molecule.

An exogenous molecule can be, among other things, a small molecule, suchas is generated by a combinatorial chemistry process, or a macromoleculesuch as a protein, nucleic acid, carbohydrate, lipid, glycoprotein,lipoprotein, polysaccharide, any modified derivative of the abovemolecules, or any complex comprising one or more of the above molecules.Nucleic acids include DNA and RNA, can be single- or double-stranded;can be linear, branched or circular; and can be of any length. Nucleicacids include those capable of forming duplexes, as well astriplex-forming nucleic acids. See, for example, U.S. Pat. Nos.5,176,996 and 5,422,251. Proteins include, but are not limited to,DNA-binding proteins, transcription factors, chromatin remodelingfactors, methylated DNA binding proteins, polymerases, methylases,demethylases, acetylases, deacetylases, kinases, phosphatases,integrases, recombinases, ligases, topoisomerases, gyrases andhelicases.

An exogenous molecule can be the same type of molecule as an endogenousmolecule, e.g., an exogenous protein or nucleic acid. For example, anexogenous nucleic acid can comprise an infecting viral genome, a plasmidor episome introduced into a cell, or a chromosome that is not normallypresent in the cell. Methods for the introduction of exogenous moleculesinto cells are known to those of skill in the art and include, but arenot limited to, lipid-mediated transfer (i.e., liposomes, includingneutral and cationic lipids), electroporation, direct injection, cellfusion, particle bombardment, calcium phosphate co-precipitation,DEAE-dextran-mediated transfer and viral vector-mediated transfer. Anexogenous molecule can also be the same type of molecule as anendogenous molecule but derived from a different species than the cellis derived from. For example, a human nucleic acid sequence may beintroduced into a cell line originally derived from a mouse or hamster.

By contrast, an “endogenous” molecule is one that is normally present ina particular cell at a particular developmental stage under particularenvironmental conditions. For example, an endogenous nucleic acid cancomprise a chromosome, the genome of a mitochondrion, chloroplast orother organelle, or a naturally-occurring episomal nucleic acid.Additional endogenous molecules can include proteins, for example,transcription factors and enzymes.

A “fusion” molecule is a molecule in which two or more subunit moleculesare linked, preferably covalently. The subunit molecules can be the samechemical type of molecule, or can be different chemical types ofmolecules. Examples of the first type of fusion molecule include, butare not limited to, fusion proteins (for example, a fusion between a ZFPor TALE DNA-binding domain and one or more activation domains) andfusion nucleic acids (for example, a nucleic acid encoding the fusionprotein described supra). Examples of the second type of fusion moleculeinclude, but are not limited to, a fusion between a triplex-formingnucleic acid and a polypeptide, and a fusion between a minor groovebinder and a nucleic acid.

Expression of a fusion protein in a cell can result from delivery of thefusion protein to the cell or by delivery of a polynucleotide encodingthe fusion protein to a cell, wherein the polynucleotide is transcribed,and the transcript is translated, to generate the fusion protein.Trans-splicing, polypeptide cleavage and polypeptide ligation can alsobe involved in expression of a protein in a cell. Methods forpolynucleotide and polypeptide delivery to cells are presented elsewherein this disclosure.

A “gene,” for the purposes of the present disclosure, includes a DNAregion encoding a gene product (see infra), as well as all DNA regionswhich regulate the production of the gene product, whether or not suchregulatory sequences are adjacent to coding and/or transcribedsequences. Accordingly, a gene includes, but is not necessarily limitedto, promoter sequences, terminators, translational regulatory sequencessuch as ribosome binding sites and internal ribosome entry sites,enhancers, silencers, insulators, boundary elements, replicationorigins, matrix attachment sites and locus control regions. An“aberrant” gene is a gene that differs in nucleotide sequence from thewild-type gene, including a gene that includes one or more mutations(e.g., point mutations, insertions and/or deletions) as compared to thewild-type sequence. Wild-type Hbb is disclosed, for example, in GenBankAccession No. NG 000007 (see, also, Efstratiadis et al. (1980) Cell21(3):653-668). The product encoded by an aberrant gene may exhibit thesame or different function (e.g., increased function, decreasedfunction, no function) as compared to the product of the wild-type gene.Exemplary aberrant Hbb genes are disclosed herein and in Kazazian andBoehm (1988) Blood vol 71 No 4: 1107.

“Gene expression” refers to the conversion of the information, containedin a gene, into a gene product. A gene product can be the directtranscriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisenseRNA, ribozyme, structural RNA or any other type of RNA) or a proteinproduced by translation of an mRNA. Gene products also include RNAswhich are modified, by processes such as capping, polyadenylation,methylation, and editing, and proteins modified by, for example,methylation, acetylation, phosphorylation, ubiquitination,ADP-ribosylation, myristilation, and glycosylation.

“Modulation” of gene expression refers to a change in the activity of agene. Modulation of expression can include, but is not limited to, geneactivation and gene repression. Genome editing (e.g., cleavage,alteration, inactivation, random mutation) can be used to modulateexpression. Gene inactivation refers to any reduction in gene expressionas compared to a cell that does not include a ZFP or TALEN as describedherein. Thus, gene inactivation may be partial or complete.

A “region of interest” is any region of cellular chromatin, such as, forexample, a gene or a non-coding sequence within or adjacent to a gene,in which it is desirable to bind an exogenous molecule. Binding can befor the purposes of targeted DNA cleavage and/or targeted recombination.A region of interest can be present in a chromosome, an episome, anorganellar genome (e.g., mitochondrial, chloroplast), or an infectingviral genome, for example. A region of interest can be within the codingregion of a gene, within transcribed non-coding regions such as, forexample, leader sequences, trailer sequences or introns, or withinnon-transcribed regions, either upstream or downstream of the codingregion. A region of interest can be as small as a single nucleotide pairor up to 2,000 nucleotide pairs in length, or any integral value ofnucleotide pairs.

“Eukaryotic” cells include, but are not limited to, fungal cells (suchas yeast), plant cells, animal cells, mammalian cells and human cells(e.g., T-cells).

“Red Blood Cells” (RBCs), or erythrocytes, are terminally differentiatedcells derived from hematopoietic stem cells. They lack a nuclease andmost cellular organelles. RBCs contain hemoglobin to carry oxygen fromthe lungs to the peripheral tissues. In fact, 33% of an individual RBCis hemoglobin. They also carry CO2 produced by cells during metabolismout of the tissues and back to the lungs for release during exhale. RBCsare produced in the bone marrow in response to blood hypoxia which ismediated by release of erythropoietin (EPO) by the kidney. EPO causes anincrease in the number of proerythroblasts and shortens the timerequired for full RBC maturation. After approximately 120 days, sincethe RBC do not contain a nucleus or any other regenerative capabilities,the cells are removed from circulation by either the phagocyticactivities of macrophages in the liver, spleen and lymph nodes (˜90%) orby hemolysis in the plasma (˜10%). Following macrophage engulfment,chemical components of the RBC are broken down within vacuoles of themacrophages due to the action of lysosomal enzymes.

“Secretory tissues” are those tissues in an animal that secrete productsout of the individual cell into a lumen of some type which are typicallyderived from epithelium. Examples of secretory tissues that arelocalized to the gastrointestinal tract include the cells that line thegut, the pancreas, and the gallbladder. Other secretory tissues includethe liver, tissues associated with the eye and mucous membranes such assalivary glands, mammary glands, the prostate gland, the pituitary glandand other members of the endocrine system. Additionally, secretorytissues include individual cells of a tissue type which are capable ofsecretion.

The terms “operative linkage” and “operatively linked” (or “operablylinked”) are used interchangeably with reference to a juxtaposition oftwo or more components (such as sequence elements), in which thecomponents are arranged such that both components function normally andallow the possibility that at least one of the components can mediate afunction that is exerted upon at least one of the other components. Byway of illustration, a transcriptional regulatory sequence, such as apromoter, is operatively linked to a coding sequence if thetranscriptional regulatory sequence controls the level of transcriptionof the coding sequence in response to the presence or absence of one ormore transcriptional regulatory factors. A transcriptional regulatorysequence is generally operatively linked in cis with a coding sequence,but need not be directly adjacent to it. For example, an enhancer is atranscriptional regulatory sequence that is operatively linked to acoding sequence, even though they are not contiguous.

With respect to fusion polypeptides, the term “operatively linked” canrefer to the fact that each of the components performs the same functionin linkage to the other component as it would if it were not so linked.For example, with respect to a fusion polypeptide in which a ZFP, TALEor Cas DNA-binding domain is fused to an activation domain, the ZFP,TALE or Cas DNA-binding domain and the activation domain are inoperative linkage if, in the fusion polypeptide, the ZFP, TALE or CasDNA-binding domain portion is able to bind its target site and/or itsbinding site, while the activation domain is able to up-regulate geneexpression. When a fusion polypeptide in which a ZFP or TALE DNA-bindingdomain is fused to a cleavage domain, the ZFP or TALE DNA-binding domainand the cleavage domain are in operative linkage if, in the fusionpolypeptide, the ZFP or TALE DNA-binding domain portion is able to bindits target site and/or its binding site, while the cleavage domain isable to cleave DNA in the vicinity of the target site.

A “functional fragment” of a protein, polypeptide or nucleic acid is aprotein, polypeptide or nucleic acid whose sequence is not identical tothe full-length protein, polypeptide or nucleic acid, yet retains thesame function as the full-length protein, polypeptide or nucleic acid. Afunctional fragment can possess more, fewer, or the same number ofresidues as the corresponding native molecule, and/or can contain one ormore amino acid or nucleotide substitutions. Methods for determining thefunction of a nucleic acid (e.g., coding function, ability to hybridizeto another nucleic acid) are well-known in the art. Similarly, methodsfor determining protein function are well-known. For example, theDNA-binding function of a polypeptide can be determined, for example, byfilter-binding, electrophoretic mobility-shift, or immunoprecipitationassays. DNA cleavage can be assayed by gel electrophoresis. See Ausubelet al., supra. The ability of a protein to interact with another proteincan be determined, for example, by co-immunoprecipitation, two-hybridassays or complementation, both genetic and biochemical. See, forexample, Fields et al. (1989) Nature 340:245-246; U.S. Pat. No.5,585,245 and PCT WO 98/44350.

A “vector” is capable of transferring gene sequences to target cells.Typically, “vector construct,” “expression vector,” and “gene transfervector,” mean any nucleic acid construct capable of directing theexpression of a gene of interest and which can transfer gene sequencesto target cells. Thus, the term includes cloning, and expressionvehicles, as well as integrating vectors.

A “reporter gene” or “reporter sequence” refers to any sequence thatproduces a protein product that is easily measured, preferably althoughnot necessarily in a routine assay. Suitable reporter genes include, butare not limited to, sequences encoding proteins that mediate antibioticresistance (e.g., ampicillin resistance, neomycin resistance, G418resistance, puromycin resistance), sequences encoding colored orfluorescent or luminescent proteins (e.g., green fluorescent protein,enhanced green fluorescent protein, red fluorescent protein,luciferase), and proteins which mediate enhanced cell growth and/or geneamplification (e.g., dihydrofolate reductase). Epitope tags include, forexample, one or more copies of FLAG, His, myc, Tap, HA or any detectableamino acid sequence. “Expression tags” include sequences that encodereporters that may be operably linked to a desired gene sequence inorder to monitor expression of the gene of interest.

The terms “subject” and “patient” are used interchangeably and refer tomammals such as human patients and non-human primates, as well asexperimental animals such as rabbits, dogs, cats, rats, mice, and otheranimals. Accordingly, the term “subject” or “patient” as used hereinmeans any mammalian patient or subject to which the altered RBCs (orstem cells) of the invention can be administered. Subjects of thepresent invention include those that have been exposed to one or morechemical toxins, including, for example, a nerve toxin.

Nucleases

Described herein are compositions, particularly nucleases, which areuseful targeting a gene for use with hemoglobinopathies. In certainembodiments, the nuclease is naturally occurring. In other embodiments,the nuclease is non-naturally occurring, i.e., engineered in theDNA-binding domain and/or cleavage domain. Any DNA-binding domain can beused in the nucleases described herein. For example, the DNA-bindingdomain of a naturally-occurring nuclease may be altered to bind to aselected target site (e.g., a meganuclease that has been engineered tobind to site different than the cognate binding site). In otherembodiments, the nuclease comprises heterologous DNA-binding andcleavage domains (e.g., zinc finger nucleases; TAL-effector nucleases;meganuclease DNA-binding domains with heterologous cleavage domains), ora generic nuclease guided by a specific guide RNA (e.g. a CRPISR/Cas).

A. DNA-Binding Domains

In certain embodiments, the nuclease is a meganuclease (homingendonuclease). Naturally-occurring meganucleases recognize 15-40base-pair cleavage sites and are commonly grouped into four families:the LAGLIDADG family, the GIY-YIG family, the His-Cyst box family andthe HNH family. Exemplary homing endonucleases include I-SceI, I-CeuI,PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII,I-CreI, I-TevI, I-TevII and I-TevIII. Their recognition sequences areknown. See also U.S. Pat. No. 5,420,032; U.S. Pat. No. 6,833,252;Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388; Dujon et al.(1989) Gene 82:115-118; Perler et al. (1994) Nucleic Acids Res. 22,1125-1127; Jasin (1996) Trends Genet. 12:224-228; Gimble et al. (1996)J. Mol. Biol. 263:163-180; Argast et al. (1998) J. Mol. Biol.280:345-353 and the New England Biolabs catalogue.

In certain embodiments, the nuclease comprises an engineered(non-naturally occurring) homing endonuclease (meganuclease). Therecognition sequences of homing endonucleases and meganucleases such asI-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII,I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIII are known. Seealso U.S. Pat. No. 5,420,032; U.S. Pat. No. 6,833,252; Belfort et al.(1997) Nucleic Acids Res. 25:3379-3388; Dujon et al. (1989) Gene82:115-118; Perler et al. (1994) Nucleic Acids Res. 22, 1125-1127; Jasin(1996) Trends Genet. 12:224-228; Gimble et al. (1996) J. Mol. Biol.263:163-180; Argast et al. (1998) J. Mol. Biol. 280:345-353 and the NewEngland Biolabs catalogue. In addition, the DNA-binding specificity ofhoming endonucleases and meganucleases can be engineered to bindnon-natural target sites. See, for example, Chevalier et al. (2002)Molec. Cell 10:895-905; Epinat et al. (2003) Nucleic Acids Res.31:2952-2962; Ashworth et al. (2006) Nature 441:656-659; Paques et al.(2007) Current Gene Therapy 7:49-66; U.S. Patent Publication No.20070117128. The DNA-binding domains of the homing endonucleases andmeganucleases may be altered in the context of the nuclease as a whole(i.e., such that the nuclease includes the cognate cleavage domain) ormay be fused to a heterologous cleavage domain.

In other embodiments, the DNA-binding domain comprises a naturallyoccurring or engineered (non-naturally occurring) TAL effector DNAbinding domain. See, e.g., U.S. Patent Publication No. 20110301073,incorporated by reference in its entirety herein. The plant pathogenicbacteria of the genus Xanthomonas are known to cause many diseases inimportant crop plants. Pathogenicity of Xanthomonas depends on aconserved type III secretion (T3S) system which injects more than 25different effector proteins into the plant cell. Among these injectedproteins are transcription activator-like effectors (TALE) which mimicplant transcriptional activators and manipulate the plant transcriptome(see Kay et at (2007) Science 318:648-651). These proteins contain a DNAbinding domain and a transcriptional activation domain. One of the mostwell characterized TALEs is AvrBs3 from Xanthomonas campestgris pv.Vesicatoria (see Bonas et at (1989) Mol Gen Genet 218: 127-136 andWO2010079430). TALEs contain a centralized domain of tandem repeats,each repeat containing approximately 34 amino acids, which are key tothe DNA binding specificity of these proteins. In addition, they containa nuclear localization sequence and an acidic transcriptional activationdomain (for a review see Schornack S, et at (2006) J Plant Physiol163(3): 256-272). In addition, in the phytopathogenic bacteria Ralstoniasolanacearum two genes, designated brg11 and hpx17 have been found thatare homologous to the AvrBs3 family of Xanthomonas in the R.solanacearum biovar 1 strain GMI1000 and in the biovar 4 strain RS1000(See Heuer et at (2007) Appl and Envir Micro 73(13): 4379-4384). Thesegenes are 98.9% identical in nucleotide sequence to each other butdiffer by a deletion of 1,575 base pairs in the repeat domain of hpx17.However, both gene products have less than 40% sequence identity withAvrBs3 family proteins of Xanthomonas.

Thus, in some embodiments, the DNA binding domain that binds to a targetsite in a target locus (e.g., globin or safe harbor) is an engineereddomain from a TAL effector similar to those derived from the plantpathogens Xanthomonas (see Boch et al, (2009) Science 326: 1509-1512 andMoscou and Bogdanove, (2009) Science326: 1501) and Ralstonia (see Heueret at (2007) Applied and Environmental Microbiology 73(13): 4379-4384);U.S. Pat. Nos. 8,420,782 and 8,440,431 and U.S. Patent Publication No.20110301073.

In certain embodiments, the DNA binding domain comprises a zinc fingerprotein (e.g., a zinc finger protein that binds to a target site in aglobin or safe-harbor gene). Preferably, the zinc finger protein isnon-naturally occurring in that it is engineered to bind to a targetsite of choice. See, for example, See, for example, Beerli et al. (2002)Nature Biotechnol. 20:135-141; Pabo et al. (2001) Ann. Rev. Biochem.70:313-340; Isalan et al. (2001) Nature Biotechnol. 19:656-660; Segal etal. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr.Opin. Struct. Biol. 10:411-416; U.S. Pat. Nos. 6,453,242; 6,534,261;6,599,692; 6,503,717; 6,689,558; 7,030,215; 6,794,136; 7,067,317;7,262,054; 7,070,934; 7,361,635; 7,253,273; and U.S. Patent PublicationNos. 2005/0064474; 2007/0218528; 2005/0267061, all incorporated hereinby reference in their entireties.

An engineered zinc finger binding or TALE domain can have a novelbinding specificity, compared to a naturally-occurring zinc fingerprotein. Engineering methods include, but are not limited to, rationaldesign and various types of selection. Rational design includes, forexample, using databases comprising triplet (or quadruplet) nucleotidesequences and individual zinc finger amino acid sequences, in which eachtriplet or quadruplet nucleotide sequence is associated with one or moreamino acid sequences of zinc fingers which bind the particular tripletor quadruplet sequence. See, for example, co-owned U.S. Pat. Nos.6,453,242 and 6,534,261, incorporated by reference herein in theirentireties.

Exemplary selection methods, including phage display and two-hybridsystems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523;6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; aswell as WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197 and GB2,338,237. In addition, enhancement of binding specificity for zincfinger binding domains has been described, for example, in co-owned WO02/077227.

In addition, as disclosed in these and other references, DNA domains(e.g., multi-fingered zinc finger proteins or TALE domains) may belinked together using any suitable linker sequences, including forexample, linkers of 5 or more amino acids in length. See, also, U.S.Pat. Nos. 6,479,626; 6,903,185; and 7,153,949 for exemplary linkersequences 6 or more amino acids in length. The DNA binding proteinsdescribed herein may include any combination of suitable linkers betweenthe individual zinc fingers of the protein. In addition, enhancement ofbinding specificity for zinc finger binding domains has been described,for example, in co-owned WO 02/077227.

Selection of target sites; DNA-binding domains and methods for designand construction of fusion proteins (and polynucleotides encoding same)are known to those of skill in the art and described in detail in U.S.Pat. Nos. 8,586,526; 7,888,121; 6,140,081; 5,789,538; 6,453,242;6,534,261; 5,925,523; 6,007,988; 6,013,453; 6,200,759.

In addition, as disclosed in these and other references, DNA-bindingdomains (e.g., multi-fingered zinc finger proteins) may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos.6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 ormore amino acids in length. The proteins described herein may includeany combination of suitable linkers between the individual zinc fingersof the protein.

In certain embodiments, the nuclease comprises a CRISPR/Cas system. TheCRISPR (clustered regularly interspaced short palindromic repeats)locus, which encodes RNA components of the system, and the cas(CRISPR-associated) locus, which encodes proteins (Jansen et al., 2002.Mol. Microbiol. 43: 1565-1575; Makarova et al., 2002. Nucleic Acids Res.30: 482-496; Makarova et al., 2006. Biol. Direct 1: 7; Haft et al.,2005. PLoS Comput. Biol. 1: e60) make up the gene sequences of theCRISPR/Cas nuclease system. CRISPR loci in microbial hosts contain acombination of CRISPR-associated (Cas) genes as well as non-coding RNAelements capable of programming the specificity of the CRISPR-mediatednucleic acid cleavage.

The Type II CRISPR is one of the most well characterized systems andcarries out targeted DNA double-strand break in four sequential steps.First, two non-coding RNA, the pre-crRNA array and tracrRNA, aretranscribed from the CRISPR locus. Second, tracrRNA hybridizes to therepeat regions of the pre-crRNA and mediates the processing of pre-crRNAinto mature crRNAs containing individual spacer sequences. Third, themature crRNA:tracrRNA complex directs Cas9 to the target DNA viaWatson-Crick base-pairing between the spacer on the crRNA and theprotospacer on the target DNA next to the protospacer adjacent motif(PAM), an additional requirement for target recognition. Finally, Cas9mediates cleavage of target DNA to create a double-stranded break withinthe protospacer. Activity of the CRISPR/Cas system comprises of threesteps: (i) insertion of alien DNA sequences into the CRISPR array toprevent future attacks, in a process called ‘adaptation’, (ii)expression of the relevant proteins, as well as expression andprocessing of the array, followed by (iii) RNA-mediated interferencewith the alien nucleic acid. Thus, in the bacterial cell, several of theso-called ‘Cas’ proteins are involved with the natural function of theCRISPR/Cas system and serve roles in functions such as insertion of thealien DNA etc.

In certain embodiments, Cas protein may be a “functional derivative” ofa naturally occurring Cas protein. A “functional derivative” of a nativesequence polypeptide is a compound having a qualitative biologicalproperty in common with a native sequence polypeptide. “Functionalderivatives” include, but are not limited to, fragments of a nativesequence and derivatives of a native sequence polypeptide and itsfragments, provided that they have a biological activity in common witha corresponding native sequence polypeptide. A biological activitycontemplated herein is the ability of the functional derivative tohydrolyze a DNA substrate into fragments. The term “derivative”encompasses both amino acid sequence variants of polypeptide, covalentmodifications, and fusions thereof. Suitable derivatives of a Caspolypeptide or a fragment thereof include but are not limited tomutants, fusions, covalent modifications of Cas protein or a fragmentthereof. Cas protein, which includes Cas protein or a fragment thereof,as well as derivatives of Cas protein or a fragment thereof, may beobtainable from a cell or synthesized chemically or by a combination ofthese two procedures. The cell may be a cell that naturally produces Casprotein, or a cell that naturally produces Cas protein and isgenetically engineered to produce the endogenous Cas protein at a higherexpression level or to produce a Cas protein from an exogenouslyintroduced nucleic acid, which nucleic acid encodes a Cas that is sameor different from the endogenous Cas. In some case, the cell does notnaturally produce Cas protein and is genetically engineered to produce aCas protein.

Exemplary CRISPR/Cas nuclease systems targeted to safe harbor and othergenes are disclosed for example, in U.S. application Ser. No.14/278,903.

In some embodiments, the DNA binding domain is part of a TtAgo system(see Swarts et al, ibid; Sheng et al, ibid). In eukaryotes, genesilencing is mediated by the Argonaute (Ago) family of proteins. In thisparadigm, Ago is bound to small (19-31 nt) RNAs. This protein-RNAsilencing complex recognizes target RNAs via Watson-Crick base pairingbetween the small RNA and the target and endonucleolytically cleaves thetarget RNA (Vogel (2014) Science 344:972-973). In contrast, prokaryoticAgo proteins bind to small single-stranded DNA fragments and likelyfunction to detect and remove foreign (often viral) DNA (Yuan et al.,(2005) Mol. Cell 19, 405; Olovnikov, et al. (2013) Mol. Cell 51, 594;Swarts et al., ibid). Exemplary prokaryotic Ago proteins include thosefrom Aquifex aeolicus, Rhodobacter sphaeroides, and Thermusthermophilus.

One of the most well-characterized prokaryotic Ago protein is the onefrom T. thermophilus (TtAgo; Swarts et al. ibid). TtAgo associates witheither 15 nt or 13-25 nt single-stranded DNA fragments with 5′ phosphategroups. This “guide DNA” bound by TtAgo serves to direct the protein-DNAcomplex to bind a Watson-Crick complementary DNA sequence in athird-party molecule of DNA. Once the sequence information in theseguide DNAs has allowed identification of the target DNA, the TtAgo-guideDNA complex cleaves the target DNA. Such a mechanism is also supportedby the structure of the TtAgo-guide DNA complex while bound to itstarget DNA (G. Sheng et al., ibid). Ago from Rhodobacter sphaeroides(RsAgo) has similar properties (Olivnikov et al. ibid).

Exogenous guide DNAs of arbitrary DNA sequence can be loaded onto theTtAgo protein (Swarts et al. ibid.). Since the specificity of TtAgocleavage is directed by the guide DNA, a TtAgo-DNA complex formed withan exogenous, investigator-specified guide DNA will therefore directTtAgo target DNA cleavage to a complementary investigator-specifiedtarget DNA. In this way, one may create a targeted double-strand breakin DNA. Use of the TtAgo-guide DNA system (or orthologous Ago-guide DNAsystems from other organisms) allows for targeted cleavage of genomicDNA within cells. Such cleavage can be either single- ordouble-stranded. For cleavage of mammalian genomic DNA, it would bepreferable to use of a version of TtAgo codon optimized for expressionin mammalian cells. Further, it might be preferable to treat cells witha TtAgo-DNA complex formed in vitro where the TtAgo protein is fused toa cell-penetrating peptide. Further, it might be preferable to use aversion of the TtAgo protein that has been altered via mutagenesis tohave improved activity at 37 degrees Celsius. Ago-RNA-mediated DNAcleavage could be used to affect a panopoly of outcomes including geneknock-out, targeted gene addition, gene correction, targeted genedeletion using techniques standard in the art for exploitation of DNAbreaks.

Thus, the nuclease can comprise any DNA-binding domain that specificallybinds to a target site in any gene.

B. Cleavage Domains

Any suitable cleavage domain can be operatively linked to anyDNA-binding domain (e.g., ZFP, TALE, guide RNA, etc.) to form a nucleaseor nuclease system. For example, ZFP, TALE, meganuclease, RNAs and otherDNA-binding domains have been fused to nuclease domains to createnucleases—a functional entity that is able to recognize its intendednucleic acid target through its engineered DNA binding domain and causethe DNA to be cut at or near the binding site via the nuclease activity.Engineered nucleases including zinc finger nucleases (“ZFNs”), TALENs,CRISPR/Cas nuclease systems, and homing endonucleases that are alldesigned to specifically bind to target DNA sites have the ability toregulate gene expression of endogenous genes and are useful in genomeengineering and gene therapy, including in the inactivation of HIVreceptors such as CCR5 and CXCR4. See, e.g., U.S. Pat. Nos. 8,586,526;6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,067,317; 7,262,054;7,888,121; 7,972,854; 7,914,796; 7,951,925; 8,110,379; 8,409,861; U.S.Patent Publications 20030232410; 20050208489; 20050026157; 20050064474;20060063231; 20080159996; 201000218264; 20120017290; 20110265198;20130137104; 20130122591; 20130177983 and 20130177960 and U.S.application Ser. No. 14/278,903, the disclosures of which are

As noted above, the cleavage domain may be heterologous to theDNA-binding domain, for example a zinc finger DNA-binding domain and acleavage domain from a nuclease or a TALEN DNA-binding domain and acleavage domain, or meganuclease DNA-binding domain and cleavage domainfrom a different nuclease. Heterologous cleavage domains can be obtainedfrom any endonuclease or exonuclease. Exemplary endonucleases from whicha cleavage domain can be derived include, but are not limited to,restriction endonucleases and homing endonucleases. See, for example,2002-2003 Catalogue, New England Biolabs, Beverly, Mass.; and Belfort etal. (1997) Nucleic Acids Res. 25:3379-3388. Additional enzymes whichcleave DNA are known (e.g., S1 Nuclease; mung bean nuclease; pancreaticDNase I; micrococcal nuclease; yeast HO endonuclease; see also Linn etal. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993). One ormore of these enzymes (or functional fragments thereof) can be used as asource of cleavage domains and cleavage half-domains.

Similarly, a cleavage half-domain can be derived from any nuclease orportion thereof, as set forth above, that requires dimerization forcleavage activity. In general, two fusion proteins are required forcleavage if the fusion proteins comprise cleavage half-domains.Alternatively, a single protein comprising two cleavage half-domains canbe used. The two cleavage half-domains can be derived from the sameendonuclease (or functional fragments thereof), or each cleavagehalf-domain can be derived from a different endonuclease (or functionalfragments thereof). In addition, the target sites for the two fusionproteins are preferably disposed, with respect to each other, such thatbinding of the two fusion proteins to their respective target sitesplaces the cleavage half-domains in a spatial orientation to each otherthat allows the cleavage half-domains to form a functional cleavagedomain, e.g., by dimerizing. Thus, in certain embodiments, the nearedges of the target sites are separated by 5-8 nucleotides or by 15-18nucleotides. However any integral number of nucleotides or nucleotidepairs can intervene between two target sites (e.g., from 2 to 50nucleotide pairs or more). In general, the site of cleavage lies betweenthe target sites.

Restriction endonucleases (restriction enzymes) are present in manyspecies and are capable of sequence-specific binding to DNA (at arecognition site), and cleaving DNA at or near the site of binding.Certain restriction enzymes (e.g., Type IIS) cleave DNA at sites removedfrom the recognition site and have separable binding and cleavagedomains. For example, the Type IIS enzyme Fok I catalyzesdouble-stranded cleavage of DNA, at 9 nucleotides from its recognitionsite on one strand and 13 nucleotides from its recognition site on theother. See, for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and5,487,994; as well as Li et al. (1992) Proc. Natl. Acad. Sci. USA89:4275-4279; Li et al. (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768;Kim et al. (1994a) Proc. Natl. Acad. Sci. USA 91:883-887; Kim et al.(1994b) J. Biol. Chem. 269:31,978-31,982. Thus, in one embodiment,fusion proteins comprise the cleavage domain (or cleavage half-domain)from at least one Type IIS restriction enzyme and one or more zincfinger binding domains, which may or may not be engineered.

An exemplary Type IIS restriction enzyme, whose cleavage domain isseparable from the binding domain, is Fok I. This particular enzyme isactive as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA95: 10,570-10,575. Accordingly, for the purposes of the presentdisclosure, the portion of the Fok I enzyme used in the disclosed fusionproteins is considered a cleavage half-domain. Thus, for targeteddouble-stranded cleavage and/or targeted replacement of cellularsequences using zinc finger-Fok I fusions, two fusion proteins, eachcomprising a FokI cleavage half-domain, can be used to reconstitute acatalytically active cleavage domain. Alternatively, a singlepolypeptide molecule containing a DNA binding domain and two Fok Icleavage half-domains can also be used.

A cleavage domain or cleavage half-domain can be any portion of aprotein that retains cleavage activity, or that retains the ability tomultimerize (e.g., dimerize) to form a functional cleavage domain.

Exemplary Type IIS restriction enzymes are described in InternationalPublication WO 07/014275, incorporated herein in its entirety.Additional restriction enzymes also contain separable binding andcleavage domains, and these are contemplated by the present disclosure.See, for example, Roberts et al. (2003) Nucleic Acids Res. 31:418-420.

In certain embodiments, the cleavage domain comprises one or moreengineered cleavage half-domain (also referred to as dimerization domainmutants) that minimize or prevent homodimerization, as described, forexample, in U.S. Pat. Nos. 7,888,121; 7,914,796; 8,034,598 and8,823,618, the disclosures of all of which are incorporated by referencein their entireties herein. Amino acid residues at positions 446, 447,479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537,and 538 of Fok I are all targets for influencing dimerization of the FokI cleavage half-domains.

Exemplary engineered cleavage half-domains of Fok I that form obligateheterodimers include a pair in which a first cleavage half-domainincludes mutations at amino acid residues at positions 490 and 538 ofFok I and a second cleavage half-domain includes mutations at amino acidresidues 486 and 499.

Thus, in one embodiment, a mutation at 490 replaces Glu (E) with Lys(K); the mutation at 538 replaces Iso (I) with Lys (K); the mutation at486 replaced Gln (Q) with Glu (E); and the mutation at position 499replaces Iso (I) with Lys (K). Specifically, the engineered cleavagehalf-domains described herein were prepared by mutating positions 490(E→K) and 538 (I→K) in one cleavage half-domain to produce an engineeredcleavage half-domain designated “E490K:1538K” and by mutating positions486 (Q→E) and 499 (I→L) in another cleavage half-domain to produce anengineered cleavage half-domain designated “Q486E:I499L”. The engineeredcleavage half-domains described herein are obligate heterodimer mutantsin which aberrant cleavage is minimized or abolished. See, e.g., U.S.Pat. No. 7,914,796, the disclosure of which is incorporated by referencein its entirety for all purposes.

In certain embodiments, the engineered cleavage half-domain comprisesmutations at positions 486, 499 and 496 (numbered relative to wild-typeFokI), for instance mutations that replace the wild type Gln (Q) residueat position 486 with a Glu (E) residue, the wild type Iso (I) residue atposition 499 with a Leu (L) residue and the wild-type Asn (N) residue atposition 496 with an Asp (D) or Glu (E) residue (also referred to as a“ELD” and “ELE” domains, respectively). In other embodiments, theengineered cleavage half-domain comprises mutations at positions 490,538 and 537 (numbered relative to wild-type FokI), for instancemutations that replace the wild type Glu (E) residue at position 490with a Lys (K) residue, the wild type Iso (I) residue at position 538with a Lys (K) residue, and the wild-type His (H) residue at position537 with a Lys (K) residue or a Arg (R) residue (also referred to as“KKK” and “KKR” domains, respectively). In other embodiments, theengineered cleavage half-domain comprises mutations at positions 490 and537 (numbered relative to wild-type FokI), for instance mutations thatreplace the wild type Glu (E) residue at position 490 with a Lys (K)residue and the wild-type His (H) residue at position 537 with a Lys (K)residue or a Arg (R) residue (also referred to as “KIK” and “KIR”domains, respectively). (See U.S. Pat. No. 8,623,618, incorporated byreference herein). Engineered cleavage half-domains described herein canbe prepared using any suitable method, for example, by site-directedmutagenesis of wild-type cleavage half-domains (Fok I) as described inU.S. Pat. Nos. 7,888,121; 7,914,796; 8,034,598 and 8,823,618.

Alternatively, nucleases may be assembled in vivo at the nucleic acidtarget site using so-called “split-enzyme” technology (see, e.g., U.S.Patent Publication No. 20090068164). Components of such split enzymesmay be expressed either on separate expression constructs, or can belinked in one open reading frame where the individual components areseparated, for example, by a self-cleaving 2A peptide or IRES sequence.Components may be individual zinc finger binding domains or domains of ameganuclease nucleic acid binding domain.

Nucleases can be screened for activity prior to use, for example in ayeast-based chromosomal system as described in WO 2009/042163 and20090068164. Nuclease expression constructs can be readily designedusing methods known in the art. See, e.g., United States PatentPublications 20030232410; 20050208489; 20050026157; 20050064474;20060188987; 20060063231; and International Publication WO 07/014275.Expression of the nuclease may be under the control of a constitutivepromoter or an inducible promoter, for example the galactokinasepromoter which is activated (de-repressed) in the presence of raffinoseand/or galactose and repressed in presence of glucose.

Target Sites

As described in detail above, DNA-binding domains can be engineered tobind to any sequence of choice in a locus, for example a globin orsafe-harbor gene. An engineered DNA-binding domain can have a novelbinding specificity, compared to a naturally-occurring DNA-bindingdomain. Engineering methods include, but are not limited to, rationaldesign and various types of selection. Rational design includes, forexample, using databases comprising triplet (or quadruplet) nucleotidesequences and individual (e.g., zinc finger) amino acid sequences, inwhich each triplet or quadruplet nucleotide sequence is associated withone or more amino acid sequences of DNA binding domain which bind theparticular triplet or quadruplet sequence. See, for example, co-ownedU.S. Pat. Nos. 6,453,242 and 6,534,261, incorporated by reference hereinin their entireties. Rational design of TAL-effector domains can also beperformed. See, e.g., U.S. Patent Publication No. 20110301073.

Exemplary selection methods applicable to DNA-binding domains, includingphage display and two-hybrid systems, are disclosed in U.S. Pat. Nos.5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466;6,200,759; and 6,242,568; as well as WO 98/37186; WO 98/53057; WO00/27878; WO 01/88197 and GB 2,338,237.

Selection of target sites; nucleases and methods for design andconstruction of fusion proteins (and polynucleotides encoding same) areknown to those of skill in the art and described in detail in U.S.Patent Application Publication Nos. 20050064474 and 20060188987,incorporated by reference in their entireties herein.

In addition, as disclosed in these and other references, DNA-bindingdomains (e.g., multi-fingered zinc finger proteins) may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids. See, e.g., U.S. Pat. Nos. 6,479,626;6,903,185; and 7,153,949 for exemplary linker sequences 6 or more aminoacids in length. The proteins described herein may include anycombination of suitable linkers between the individual DNA-bindingdomains of the protein. See, also, U.S. Patent Publication No.20110287512.

Donors

As noted above, insertion of an exogenous sequence (also called a “donorsequence” or “donor” or “transgene”), for example for correction of amutant gene or for increased expression of a wild-type gene. It will bereadily apparent that the donor sequence is typically not identical tothe genomic sequence where it is placed. A donor sequence can contain anon-homologous sequence flanked by two regions of homology to allow forefficient HDR at the location of interest. Additionally, donor sequencescan comprise a vector molecule containing sequences that are nothomologous to the region of interest in cellular chromatin. A donormolecule can contain several, discontinuous regions of homology tocellular chromatin. For example, for targeted insertion of sequences notnormally present in a region of interest, said sequences can be presentin a donor nucleic acid molecule and flanked by regions of homology tosequence in the region of interest.

Described herein are methods of targeted insertion of anypolynucleotides for insertion into a chosen location. Polynucleotidesfor insertion can also be referred to as “exogenous” polynucleotides,“donor” polynucleotides or molecules or “transgenes.” The donorpolynucleotide can be DNA or RNA, single-stranded and/or double-strandedand can be introduced into a cell in linear or circular form. See, e.g.,U.S. Patent Publication Nos. 20100047805, 20110281361, 20110207221 andU.S. application Ser. No. 13/889,162. The donor sequence(s) can becontained within a DNA MC, which may be introduced into the cell incircular or linear form. If introduced in linear form, the ends of thedonor sequence can be protected (e.g., from exonucleolytic degradation)by methods known to those of skill in the art. For example, one or moredideoxynucleotide residues are added to the 3′ terminus of a linearmolecule and/or self-complementary oligonucleotides are ligated to oneor both ends. See, for example, Chang et al. (1987) Proc. Natl. Acad.Sci. USA 84:4959-4963; Nehls et al. (1996) Science 272:886-889.Additional methods for protecting exogenous polynucleotides fromdegradation include, but are not limited to, addition of terminal aminogroup(s) and the use of modified internucleotide linkages such as, forexample, phosphorothioates, phosphoramidates, and O-methyl ribose ordeoxyribose residues. If introduced in double-stranded form, the donormay include one or more nuclease target sites, for example, nucleasetarget sites flanking the transgene to be integrated into the cell'sgenome. See, e.g., U.S. Patent Publication No. 20130326645.

A polynucleotide can be introduced into a cell as part of a vectormolecule having additional sequences such as, for example, replicationorigins, promoters and genes encoding antibiotic resistance. Moreover,donor polynucleotides can be introduced as naked nucleic acid, asnucleic acid complexed with an agent such as a liposome or poloxamer, orcan be delivered by viruses (e.g., adenovirus, AAV, herpesvirus,retrovirus, lentivirus and integrase defective lentivirus (IDLV)).Suitable non-viral vectors include nanotaxis vectors, including vectorscommercially available from InCellArt (France).

In certain embodiments, the double-stranded donor includes sequences(e.g., coding sequences, also referred to as transgenes) greater than 1kb in length, for example between 2 and 200 kb, between 2 and 10 kb (orany value therebetween). The double-stranded donor also includes atleast one nuclease target site, for example. In certain embodiments, thedonor includes at least 1 target site, for example, for use with aCRISPR/Cas, or 2 target sites, for example for a pair of ZFNs or TALENs.Typically, the nuclease target sites are outside the transgenesequences, for example, 5′ and/or 3′ to the transgene sequences, forcleavage of the transgene. The nuclease cleavage site(s) may be for anynuclease(s). In certain embodiments, the nuclease target site(s)contained in the double-stranded donor are for the same nuclease(s) usedto cleave the endogenous target into which the cleaved donor isintegrated via homology-independent methods.

The donor is generally inserted so that its expression is driven by theendogenous promoter at the integration site, namely the promoter thatdrives expression of the endogenous gene into which the donor isinserted (e.g., globin, AAVS1, etc.). However, it will be apparent thatthe donor may comprise a promoter and/or enhancer, for example aconstitutive promoter or an inducible or tissue specific promoter.

The donor molecule may be inserted into an endogenous gene such thatall, some or none of the endogenous gene is expressed. For example, atransgene as described herein may be inserted into a globin locus suchthat some or none of the endogenous globin sequences are expressed, forexample as a fusion with the transgene. In other embodiments, thetransgene (e.g., with or without globin encoding sequences) isintegrated into any endogenous locus, for example a safe-harbor locus.See, e.g., US patent publications 20080299580; 20080159996 and201000218264.

When additional (e.g., globin sequences, endogenous or part of thetransgene) are expressed with the transgene, the additionally (e.g.,globin) sequences may be full-length sequences (wild-type or mutant) orpartial sequences. Preferably, the additional sequences are functional.Non-limiting examples of the function of these full length or partialadditional sequences, for example globin-encoding sequences, includeincreasing the serum half-life of the polypeptide expressed by thetransgene (e.g., therapeutic gene) and/or acting as a carrier.

Furthermore, although not required for expression, exogenous sequencesmay also include transcriptional or translational regulatory sequences,for example, promoters, enhancers, insulators, internal ribosome entrysites, sequences encoding 2A peptides and/or polyadenylation signals.

The transgenes carried on the donor sequences described herein may beisolated from plasmids, cells or other sources using standard techniquesknown in the art such as PCR. Donors for use can include varying typesof topology, including circular supercoiled, circular relaxed, linearand the like. Alternatively, they may be chemically synthesized usingstandard oligonucleotide synthesis techniques. In addition, donors maybe methylated or lack methylation. Donors may be in the form ofbacterial or yeast artificial chromosomes (BACs or YACs).

The double-stranded donor polynucleotides described herein may includeone or more non-natural bases and/or backbones. In particular, insertionof a donor molecule with methylated cytosines may be carried out usingthe methods described herein to achieve a state of transcriptionalquiescence in a region of interest.

The exogenous (donor) polynucleotide may comprise any sequence ofinterest (exogenous sequence). Exemplary exogenous sequences include,but are not limited to any polypeptide coding sequence (e.g., cDNAs),promoter sequences, enhancer sequences, epitope tags, marker genes,cleavage enzyme recognition sites and various types of expressionconstructs. Marker genes include, but are not limited to, sequencesencoding proteins that mediate antibiotic resistance (e.g., ampicillinresistance, neomycin resistance, G418 resistance, puromycin resistance),sequences encoding colored or fluorescent or luminescent proteins (e.g.,green fluorescent protein, enhanced green fluorescent protein, redfluorescent protein, luciferase), and proteins which mediate enhancedcell growth and/or gene amplification (e.g., dihydrofolate reductase).Epitope tags include, for example, one or more copies of FLAG, His, myc,Tap, HA or any detectable amino acid sequence.

In a preferred embodiment, the exogenous sequence (transgene) comprisesa polynucleotide encoding any polypeptide of which expression in thecell is desired, including, but not limited to antibodies, antigens,enzymes, receptors (cell surface or nuclear), hormones, lymphokines,cytokines, reporter polypeptides, growth factors, and functionalfragments of any of the above. The coding sequences may be, for example,cDNAs.

In certain embodiments, the exogenous sequences can comprise a markergene (described above), allowing selection of cells that have undergonetargeted integration, and a linked sequence encoding an additionalfunctionality. Non-limiting examples of marker genes include GFP, drugselection marker(s) and the like.

Additional gene sequences that can be inserted may include, for example,wild-type genes to replace mutated sequences. For example, a wild-typebeta globin gene sequence may be inserted into the genome of a stem cellin which the endogenous copy of the gene is mutated. The wild-type copymay be inserted at the endogenous locus, or may alternatively betargeted to a safe harbor locus.

Construction of such expression cassettes, following the teachings ofthe present specification, utilizes methodologies well known in the artof molecular biology (see, for example, Ausubel or Maniatis). Before useof the expression cassette to generate a transgenic animal, theresponsiveness of the expression cassette to the stress-inducerassociated with selected control elements can be tested by introducingthe expression cassette into a suitable cell line (e.g., primary cells,transformed cells, or immortalized cell lines).

Furthermore, although not required for expression, exogenous sequencesmay also transcriptional or translational regulatory sequences, forexample, promoters, enhancers, insulators, internal ribosome entrysites, sequences encoding 2A peptides and/or polyadenylation signals.Further, the control elements of the genes of interest can be operablylinked to reporter genes to create chimeric genes (e.g., reporterexpression cassettes).

Targeted insertion of non-coding nucleic acid sequence may also beachieved. Sequences encoding antisense RNAs, RNAi, shRNAs and micro RNAs(miRNAs) may also be used for targeted insertions.

In additional embodiments, the donor nucleic acid may comprisenon-coding sequences that are specific target sites for additionalnuclease designs. Subsequently, additional nucleases may be expressed incells such that the original donor molecule is cleaved and modified byinsertion of another donor molecule of interest. In this way,reiterative integrations of donor molecules may be generated allowingfor trait stacking at a particular locus of interest or at a safe harborlocus.

Delivery

The nucleases, polynucleotides encoding these nucleases, donorpolynucleotides and compositions comprising the proteins and/orpolynucleotides described herein may be delivered in vivo or ex vivo byany suitable means.

Methods of delivering nucleases as described herein are described, forexample, in U.S. Pat. Nos. 6,453,242; 6,503,717; 6,534,261; 6,599,692;6,607,882; 6,689,558; 6,824,978; 6,933,113; 6,979,539; 7,013,219; and7,163,824, the disclosures of all of which are incorporated by referenceherein in their entireties.

Nucleases and/or donor constructs as described herein may also bedelivered using vectors containing sequences encoding one or more of thezinc finger or TALEN protein(s). Any vector systems may be usedincluding, but not limited to, plasmid vectors, retroviral vectors,lentiviral vectors, adenovirus vectors, poxvirus vectors; herpesvirusvectors and adeno-associated virus vectors, etc. See, also, U.S. Pat.Nos. 6,534,261; 6,607,882; 6,824,978; 6,933,113; 6,979,539; 7,013,219;and 7,163,824, incorporated by reference herein in their entireties.Furthermore, it will be apparent that any of these vectors may compriseone or more of the sequences needed for treatment. Thus, when one ormore nucleases and a donor construct are introduced into the cell, thenucleases and/or donor polynucleotide may be carried on the same vectoror on different vectors. When multiple vectors are used, each vector maycomprise a sequence encoding one or multiple nucleases and/or donorconstructs.

Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids encoding nucleases and donor constructs incells (e.g., mammalian cells) and target tissues. Non-viral vectordelivery systems include DNA plasmids, naked nucleic acid, and nucleicacid complexed with a delivery vehicle such as a liposome or poloxamer.Viral vector delivery systems include DNA and RNA viruses, which haveeither episomal or integrated genomes after delivery to the cell. For areview of gene therapy procedures, see Anderson, Science 256:808-813(1992); Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani & Caskey,TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller,Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10):1149-1154(1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995);Kremer & Perricaudet, British Medical Bulletin 51(1):31-44 (1995);Haddada et al., in Current Topics in Microbiology and ImmunologyDoerfler and Bohm (eds.) (1995); and Yu et al., Gene Therapy 1:13-26(1994).

Methods of non-viral delivery of nucleic acids include electroporation,lipofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,artificial virions, and agent-enhanced uptake of DNA. Sonoporationusing, e.g., the Sonitron 2000 system (Rich-Mar) can also be used fordelivery of nucleic acids.

Additional exemplary nucleic acid delivery systems include thoseprovided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc.(Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.) andCopernicus Therapeutics Inc., (see for example U.S. Pat. No. 6,008,336).Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386; 4,946,787;and 4,897,355) and lipofection reagents are sold commercially (e.g.,Transfectam™ and Lipofectin™). Cationic and neutral lipids that aresuitable for efficient receptor-recognition lipofection ofpolynucleotides include those of Felgner, WO 91/17424, WO 91/16024.

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

Additional methods of delivery include the use of packaging the nucleicacids to be delivered into EnGeneIC delivery vehicles (EDVs). These EDVsare specifically delivered to target tissues using bispecific antibodieswhere one arm of the antibody has specificity for the target tissue andthe other has specificity for the EDV. The antibody brings the EDVs tothe target cell surface and then the EDV is brought into the cell byendocytosis. Once in the cell, the contents are released (see MacDiarmidet at (2009) Nature Biotechnology 27(7):643).

The use of RNA or DNA viral based systems for the delivery of nucleicacids encoding engineered ZFPs take advantage of highly evolvedprocesses for targeting a virus to specific cells in the body andtrafficking the viral payload to the nucleus. Viral vectors can beadministered directly to subjects (in vivo) or they can be used to treatcells in vitro and the modified cells are administered to subjects (exvivo). Conventional viral based systems for the delivery of ZFPsinclude, but are not limited to, retroviral, lentivirus, adenoviral,adeno-associated, vaccinia and herpes simplex virus vectors for genetransfer. Integration in the host genome is possible with theretrovirus, lentivirus, and adeno-associated virus gene transfermethods, often resulting in long term expression of the insertedtransgene. Additionally, high transduction efficiencies have beenobserved in many different cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system depends on thetarget tissue. Retroviral vectors are comprised of cis-acting longterminal repeats with packaging capacity for up to 6-10 kb of foreignsequence. The minimum cis-acting LTRs are sufficient for replication andpackaging of the vectors, which are then used to integrate thetherapeutic gene into the target cell to provide permanent transgeneexpression. Widely used retroviral vectors include those based uponmurine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), SimianImmunodeficiency virus (SW), human immunodeficiency virus (HIV), andcombinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700).

In applications in which transient expression is preferred, adenoviralbased systems can be used. Adenoviral based vectors are capable of veryhigh transduction efficiency in many cell types and do not require celldivision. With such vectors, high titer and high levels of expressionhave been obtained. This vector can be produced in large quantities in arelatively simple system. Adeno-associated virus (“AAV”) vectors arealso used to transduce cells with target nucleic acids, e.g., in the invitro production of nucleic acids and peptides, and for in vivo and exvivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47(1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994).Construction of recombinant AAV vectors are described in a number ofpublications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol.Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol.4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); andSamulski et al., J. Virol. 63:03822-3828 (1989).

At least six viral vector approaches are currently available for genetransfer in clinical trials, which utilize approaches that involvecomplementation of defective vectors by genes inserted into helper celllines to generate the transducing agent.

pLASN and MFG-S are examples of retroviral vectors that have been usedin clinical trials (Dunbar et al., Blood 85:3048-305 (1995); Kohn etal., Nat. Med. 1:1017-102 (1995); Malech et al., PNAS 94:22 12133-12138(1997)). PA317/pLASN was the first therapeutic vector used in a genetherapy trial. (Blaese et al., Science 270:475-480 (1995)). Transductionefficiencies of 50% or greater have been observed for MFG-S packagedvectors. (Ellem et al., Immunol Immunother. 44(1):10-20 (1997); Dranoffet al., Hum. Gene Ther. 1:111-2 (1997).

Recombinant adeno-associated virus vectors (rAAV) are a promisingalternative gene delivery systems based on the defective andnonpathogenic parvovirus adeno-associated type 2 virus. All vectors arederived from a plasmid that retains only the AAV 145 base pair invertedterminal repeats flanking the transgene expression cassette. Efficientgene transfer and stable transgene delivery due to integration into thegenomes of the transduced cell are key features for this vector system.(Wagner et al., Lancet 351:9117 1702-3 (1998), Kearns et al., Gene Ther.9:748-55 (1996)). Other AAV serotypes, including AAV1, AAV3, AAV4, AAV5,AAV6, AAV8, AAV9 and AAVrh10, and all variants thereof, can also be usedin accordance with the present invention.

Replication-deficient recombinant adenoviral vectors (Ad) can beproduced at high titer and readily infect a number of different celltypes. Most adenovirus vectors are engineered such that a transgenereplaces the Ad E1a, E1b, and/or E3 genes; subsequently the replicationdefective vector is propagated in human 293 cells that supply deletedgene function in trans. Ad vectors can transduce multiple types oftissues in vivo, including non-dividing, differentiated cells such asthose found in liver, kidney and muscle. Conventional Ad vectors have alarge carrying capacity. An example of the use of an Ad vector in aclinical trial involved polynucleotide therapy for anti-tumorimmunization with intramuscular injection (Sterman et al., Hum. GeneTher. 7:1083-9 (1998)). Additional examples of the use of adenovirusvectors for gene transfer in clinical trials include Rosenecker et al.,Infection 24:1 5-10 (1996); Sterman et al., Hum. Gene Ther. 9:71083-1089 (1998); Welsh et al., Hum. Gene Ther. 2:205-18 (1995); Alvarezet al., Hum. Gene Ther. 5:597-613 (1997); Topf et al., Gene Ther.5:507-513 (1998); Sterman et al., Hum. Gene Ther. 7:1083-1089 (1998).

Packaging cells are used to form virus particles that are capable ofinfecting a host cell. Such cells include 293 cells, which packageadenovirus, and ψ2 cells or PA317 cells, which package retrovirus. Viralvectors used in gene therapy are usually generated by a producer cellline that packages a nucleic acid vector into a viral particle. Thevectors typically contain the minimal viral sequences required forpackaging and subsequent integration into a host (if applicable), otherviral sequences being replaced by an expression cassette encoding theprotein to be expressed. The missing viral functions are supplied intrans by the packaging cell line. For example, AAV vectors used in genetherapy typically only possess inverted terminal repeat (ITR) sequencesfrom the AAV genome which are required for packaging and integrationinto the host genome. Viral DNA is packaged in a cell line, whichcontains a helper plasmid encoding the other AAV genes, namely rep andcap, but lacking ITR sequences. The cell line is also infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV.

In many gene therapy applications, it is desirable that the gene therapyvector be delivered with a high degree of specificity to a particulartissue type. Accordingly, a viral vector can be modified to havespecificity for a given cell type by expressing a ligand as a fusionprotein with a viral coat protein on the outer surface of the virus. Theligand is chosen to have affinity for a receptor known to be present onthe cell type of interest. For example, Han et al., Proc. Natl. Acad.Sci. USA 92:9747-9751 (1995), reported that Moloney murine leukemiavirus can be modified to express human heregulin fused to gp70, and therecombinant virus infects certain human breast cancer cells expressinghuman epidermal growth factor receptor. This principle can be extendedto other virus-target cell pairs, in which the target cell expresses areceptor and the virus expresses a fusion protein comprising a ligandfor the cell-surface receptor. For example, filamentous phage can beengineered to display antibody fragments (e.g., FAB or Fv) havingspecific binding affinity for virtually any chosen cellular receptor.Although the above description applies primarily to viral vectors, thesame principles can be applied to nonviral vectors. Such vectors can beengineered to contain specific uptake sequences which favor uptake byspecific target cells.

Gene therapy vectors can be delivered in vivo by administration to anindividual subject, typically by systemic administration (e.g.,intravenous, intraperitoneal, intramuscular, subdermal, or intracranialinfusion) or topical application, as described below. Alternatively,vectors can be delivered to cells ex vivo, such as cells explanted froman individual patient (e.g., lymphocytes, bone marrow aspirates, tissuebiopsy) or universal donor hematopoietic stem cells, followed byreimplantation of the cells into a patient, usually after selection forcells which have incorporated the vector.

Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containingnucleases and/or donor constructs can also be administered directly toan organism for transduction of cells in vivo. Alternatively, naked DNAcan be administered. Administration is by any of the routes normallyused for introducing a molecule into ultimate contact with blood ortissue cells including, but not limited to, injection, infusion, topicalapplication and electroporation. Suitable methods of administering suchnucleic acids are available and well known to those of skill in the art,and, although more than one route can be used to administer a particularcomposition, a particular route can often provide a more immediate andmore effective reaction than another route.

Vectors suitable for introduction of polynucleotides described hereininclude non-integrating lentivirus vectors (IDLV). See, for example, Oryet al. (1996) Proc. Natl. Acad. Sci. USA 93:11382-11388; Dull et al.(1998) J. Virol. 72: 8463-8471; Zuffery et al. (1998) J. Virol.72:9873-9880; Follenzi et al. (2000) Nature Genetics 25:217-222; U.S.Patent Publication No 20090117617.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositionsavailable, as described below (see, e.g., Remington's PharmaceuticalSciences, 17th ed., 1989).

It will be apparent that the nuclease-encoding sequences and donorconstructs can be delivered using the same or different systems. Forexample, a donor polynucleotide can be carried by a plasmid, while theone or more nucleases can be carried by an AAV vector. Furthermore, thedifferent vectors can be administered by the same or different routes(intramuscular injection, tail vein injection, other intravenousinjection, intraperitoneal administration and/or intramuscularinjection. The vectors can be delivered simultaneously or in anysequential order.

Thus, the instant disclosure includes in vivo or ex vivo treatment ofdiseases and conditions that are amenable to insertion of a transgenesencoding a therapeutic protein, for example treatment ofhemoglobinopathies via nuclease-mediated integration of a gene encodinga globin protein. The compositions are administered to a human patientin an amount effective to obtain the desired concentration of thetherapeutic polypeptide in the serum or the target organ or cells.Administration can be by any means in which the polynucleotides aredelivered to the desired target cells. For example, both in vivo and exvivo methods are contemplated. Intravenous injection to the portal veinis a preferred method of administration. Other in vivo administrationmodes include, for example, direct injection into the lobes of the liveror the biliary duct and intravenous injection distal to the liver,including through the hepatic artery, direct injection in to the liverparenchyma, injection via the hepatic artery, and/or retrogradeinjection through the biliary tree. Ex vivo modes of administrationinclude transduction in vitro of resected hepatocytes or other cells ofthe liver, followed by infusion of the transduced, resected hepatocytesback into the portal vasculature, liver parenchyma or biliary tree ofthe human patient, see e.g., Grossman et al., (1994) Nature Genetics,6:335-341.

The effective amount of nuclease(s) and donor to be administered willvary from patient to patient and according to the therapeuticpolypeptide of interest. Accordingly, effective amounts are bestdetermined by the physician administering the compositions andappropriate dosages can be determined readily by one of ordinary skillin the art. After allowing sufficient time for integration andexpression (typically 4-15 days, for example), analysis of the serum orother tissue levels of the therapeutic polypeptide and comparison to theinitial level prior to administration will determine whether the amountbeing administered is too low, within the right range or too high.Suitable regimes for initial and subsequent administrations are alsovariable, but are typified by an initial administration followed bysubsequent administrations if necessary. Subsequent administrations maybe administered at variable intervals, ranging from daily to annually toevery several years. One of skill in the art will appreciate thatappropriate immunosuppressive techniques may be recommended to avoidinhibition or blockage of transduction by immunosuppression of thedelivery vectors, see e.g., Vilquin et al., (1995) Human Gene Ther.6:1391-1401.

Formulations for both ex vivo and in vivo administrations includesuspensions in liquid or emulsified liquids. The active ingredientsoften are mixed with excipients which are pharmaceutically acceptableand compatible with the active ingredient. Suitable excipients include,for example, water, saline, dextrose, glycerol, ethanol or the like, andcombinations thereof. In addition, the composition may contain minoramounts of auxiliary substances, such as, wetting or emulsifying agents,pH buffering agents, stabilizing agents or other reagents that enhancethe effectiveness of the pharmaceutical composition.

Cells

Also described herein are cells and/or cell lines in which an endogenoussequence human beta-hemoglobin (Hbb) is modified. The modification maybe, for example, as compared to the wild-type sequence of the cell,which may be a cell from a subject with a thalassemia. The cell or celllines may be heterozygous or homozygous for the modification. Themodifications are may comprise insertions, deletions and/or combinationsthereof.

The Hbb gene may be modified by a nuclease (e.g., ZFN, TALEN, CRISPR/Cassystem, Ttago system, etc.), for example a nuclease as described herein.In certain embodiments, the genomic modification is to an interveningsequence 1, mutation number 1 (IVS1-1 or IVS.1), IVS1-5, IVS1-6,IVS1-110, IVS-1, IVS2-745 or IVS2-654 mutation, for example amodification that corrects a mutant IVS.1 sequence as seen in subjectswith a thalassemia such as beta-thalassemia. In certain embodiments, theinvention comprises the genetically modified cell described above,wherein the genomic modification is within one or more of the sequencesshown in SEQ ID NO:3, for example that disrupts a splice donor motif(e.g., modification of a “GT” dinucleotide at the beginning of intron 1of the human beta-globin gene is modified to an “AT”). In certainembodiments, the genomic modification corrects a mutation that altersthe splice donor site for mRNA processing of the Hbb gene and leads tobeta-thalassemia.

The modification is preferably at or near the nuclease(s) binding and/orcleavage site(s), for example, within 1-300 (or any value therebetween)base pairs upstream or downstream of the site(s) of cleavage, morepreferably within 1-100 base pairs (or any value therebetween) of eitherside of the binding and/or cleavage site(s), even more preferably within1 to 50 base pairs (or any value therebetween) on either side of thebinding and/or cleavage site(s). In certain embodiments, themodification is at or near any target sequence shown in SEQ ID NO:3, 4or 5.

Any cell or cell line may be modified, for example a stem cell, forexample an embryonic stem cell, an induced pluripotent stem cell, ahematopoietic stem cell, a neuronal stem cell and a mesenchymal stemcell. Other non-limiting examples of cells as described herein includeT-cells (e.g., CD4+, CD3+, CD8+, etc.); dendritic cells; B-cells. Adescendent of a stem cell, including a partially or fully differentiatedcell, is also provided (e.g., a RBC or RBC precursor cell). Non-limitingexamples other cell lines including a modified BCL11A sequence includeCOS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44, CHO-DUXB11, CHO-DUKX, CHOK1SV),VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NS0, SP2/0-Ag14, HeLa,HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T), and perC6 cells as well asinsect cells such as Spodopterafugiperda (Sf), or fungal cells such asSaccharomyces, Pichia and Schizosaccharomyces.

The cells as described herein are useful in treating and/or preventing adisorder, for example, by ex vivo therapies. The nuclease-modified cellscan be expanded and then reintroduced into the patient using standardtechniques. See, e.g., Tebas et at (2014) New Eng J Med 370(10):901. Inthe case of stem cells, after infusion into the subject, in vivodifferentiation of these precursors into cells expressing the functionaltransgene also occurs. Pharmaceutical compositions comprising the cellsas described herein are also provided. In addition, the cells may becryopreserved prior to administration to a patient.

Any of the modified cells or cell lines disclosed herein may showincreased expression of a wild-type or inserted. Compositions such aspharmaceutical compositions comprising the genetically modified cells asdescribed herein are also provided

Applications

The methods and compositions disclosed herein are for modifyingexpression of protein, or correcting an aberrant gene sequence in agenetic disease, such as a thalassemia. Thus, the methods andcompositions provide for the treatment and/or prevention of such geneticdiseases. Genome editing, for example of stem cells, is used to correctan aberrant gene, insert a wild type gene, or change the expression ofan endogenous gene. By way of non-limiting example, a wild type gene,e.g. encoding at least one globin (e.g. β globin), may be inserted intoa cell to provide the globin proteins deficient and/or lacking in thecell and thereby treat a genetic disease, e.g., a hemoglobinopathy.Alternatively or in addition, genomic editing with or withoutadministration of the appropriate donor, can correct the faultyendogenous gene, e.g., correcting the point mutation in β-hemoglobin, torestore expression of the gene and/or treat a genetic disease, e.g.beta-thalassemia.

The following Examples relate to exemplary embodiments of the presentdisclosure in which the nuclease comprises a zinc finger nuclease (ZFN).It will be appreciated that this is for purposes of exemplification onlyand that other nucleases can be used, for instance TALENs, homingendonucleases (meganucleases) with engineered DNA-binding domains and/orfusions of naturally occurring of engineered homing endonucleases(meganucleases) and DNA-binding domains and heterologous cleavagedomains, (e.g. Mega-TAL) and/or a CRISPR/Cas system comprising anengineered single guide RNA.

EXAMPLES Example 1 Design, Construction and General Characterization ofZinc Finger Protein Nucleases (ZFN)

Zinc finger proteins were designed and incorporated into plasmids, AAVor adenoviral vectors essentially as described in Urnov et al. (2005)Nature 435(7042):646-651, Perez et at (2008) Nature Biotechnology26(7):808-816, and as described in U.S. Pat. No. 6,534,261. For ZFNs andTALENs specific for the human beta globin locus see, also, co-owned U.S.Pat. No. 7,888,121.

Example 2 Activity of Beta Globin-Specific ZFNs

ZFN pairs targeting the human beta globin locus at or near the IVS1-1mutation were made as described above. See, also, U.S. PatentPublication No. 20140093913. The amino acid sequences of the recognitionhelix regions of each finger of the indicated ZFNs are shown below inTable 1 along with the whole target sites (DNA target sites indicated inuppercase letters; non-contacted nucleotides indicated in lowercase).The location of the target site is also shown in FIG. 1. All ZFPs weretested for binding specificity to their target sequence.

TABLE 1 Zinc finger nucleases SBS #, Target DesignHuman B-Hemoglobin IVS1-1 region specific ZFNs F1 F2 F3 F4 F5 F6SBS#43544 AMQTLRV DRSHLAR RSDNLSE ASKTRKN RNSDRTK N/A aTCTGCCCAGGGC(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID CTCaccaccaact NO: 6) NO: 7)NO: 8) NO: 9) NO: 10) t (SEQ ID NO: 4) SBS#43545 LRHHLTR QSGTRKT RSDNLSTDSANRIK LRHHLTR QSGNLHV atCAAGGTTACAA  (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID GACAGGTttaagg NO: 11) NO: 12) NO: 13) NO: 14) NO: 11)NO: 15) ag (SEQ ID NO: 5) SBS#45946 AMQTLRV DRSHLAR RSDNLSE ASKTRKNTSSDRKK N/A aaTCTGCCCAGGG (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ IDCCTCaccaccaac NO: 16) NO: 7) NO: 8) NO: 9) NO: 17) tt (SEQ ID NO: 4)SBS#45952 AMQTLRV DRSHLAR RSDNLSE ASKTRKN VYEGLKK N/A aaTCTGCCCAGGG(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID CCTCaccaccaac NO: 16) NO: 7)NO: 8) NO: 9) NO: 18) tt (SEQ ID NO: 4)

The Cel-I assay (Surveyor™, Transgenomics) as described in Perez et al.(2008) Nat. Biotechnol. 26: 808-816 and Guschin et al. (2010) MethodsMol Biol. 649:247-56), was used to detect ZFN-induced modifications ofthe target gene in K562 or HSCs. In this assay, PCR-amplification of thetarget site was followed by quantification of insertions and/ordeletions (“indels”) using the mismatch detecting enzyme Cel-I (Yang etal. (2000) Biochemistry 39: 3533-3541) which provided a lower-limitestimate of DSB frequency. Three days following transfection of the ZFNexpression vector at either standard conditions (37° C.) or using ahypothermic shock (30° C., see co-owned US patent publication U.S.Patent Publication No. 20110041195), genomic DNA was isolated from K562cells using the DNeasy kit (Qiagen).

The results (FIG. 2A) from the Cel-I assay demonstrated that the ZFNswere capable of inducing cleavage at their respective target sites. Inaddition, the ZFNs were tested for activity using the Dual-LuciferaseSingle Strand Annealing Assay (DLSSA). This system was used to quantifyZFN activity in transiently transfected cells, and is based on theDual-Luciferase Reporter® Assay System from Promega. The systemsequential measures two individual reporter enzymes, Firefly and RenillaLuciferases, within a single tube (well). Both of the Firefly and theRenilla Luciferase reporters are re-engineered and the assay conditionsare optimized. The Firefly Luciferase reporter construct contains twoincomplete copies of the Firefly coding regions that are separated byDNA binding sites for the ZFNs. In this study, the 5′ copy is derivedfrom approximately two third of the N-terminal part of the Firefly geneand the 3′ copy is derived from approximately two third of theC-terminal part of the Firefly gene. The two incomplete copies containabout 600-bp homology arms. The separated Firefly fragments have noluciferase activity. A DNA double strand break caused by the ZFN pairwill stimulate recombination between flanking repeats by thesingle-strand annealing pathway and then restore the Firefly luciferasefunction. The co-transfected Renilla Luciferase plasmid provides aninternal control. The luminescent activity of each reporter is read on aluminometer. Normalizing the activity of the experimental reporter(Firefly) to the activity of the internal control (Renilla) minimizesexperimental variability caused by differences in cell viability and/ortransfection efficiency. The normalized value is used to determine theactivity of a given ZFN or TALEN pair.

The results (FIG. 2B) demonstrate that the 43545/43544 pair is capableof cleaving both the wild type beta globin sequence and the mutant IVS1-1 sequence.

Example 3 Editing of the IVS 1-1 Beta Globin Locus

The human beta globin gene (HBB) specific ZFN 43545/43544 pair was usedto introduce a donor DNA into the beta globin locus as follows. A donoroligo was designed for capture into the cleaved HBB gene following ZFNtreatment. The oligo contained an XbaI restriction site such thatfollowing insertion of the oligo, a novel restriction site wasintroduced into the HBB gene intron (IVS1) right after the ZFNrecognition site that could subsequently be visualized by restrictiondigest of a PCR product containing the ZFN binding region (see, FIG. 3).

The various oligos were delivered to CD34+ cells as single strandedmolecules and are shown below:

Hbb oligo WT: (SEQ ID NO: 19) 5′AAGTCTGCCGTTACTGCCCTGTGGGGCAAGGTGAACGTGGATGAAGTTGGTGGTGAGGCCCTGGGCAGGTTGGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCATGTGGAGACAGA Hbb oligo G_A: (SEQ ID NO: 20) 5′AAGTCTGCCGTTACTGCCCTGTGGGGCAAGGTGAACGTGGATGAAGTTGGTGGTGAGGCCCTGGGCAGATTGGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCATGTGGAGACAGA Hbb oligo WT XbaI: (SEQ ID NO: 21) 5′AAGTCTGCCGTTACTGCCCTGTGGGGCAAGGTGAACGTGGATGAAGTTGGTGGTGAGGCCCTGGGCAGGTTGGTATCTAGAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCATGTGGAGACAGA Hbb oligo G_A XbaI: (SEQ ID NO: 22)5′ AAGTCTGCCGTTACTGCCCTGTGGGGCAAGGTGAACGTGGATGAAGTTGGTGGTGAGGCCCTGGGCAGATTGGTATCTAGAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCATGTGGAGACAGA

For the CD34+ transduction, a BTX ECM830 device with a 2 mm gap cuvettewas used. mRNAs from the cells were prepared using a mMessageMachine T7Ultra Kit (#AM1345, Ambion). Human CD34+ cells were grown in x-vivo10media (Lonza) with 1xCC110 (Stem cell Technology) in non-tissue culturetreated plates. The cells were counted and collected by centrifugationat 1200 rpm for 10 minutes at room temperature. The cells were washed1-2× with room temperature PBS. 200,000 cells were used for eachtransfection, and they were resuspended in 100 μL BTexpress solution.2-4 μg mRNA was added per transfection and the mixture was transferredto the cuvette. Immediately following transfer, the mixture waselectroporated at 250V for 5 msec. Pre-warmed media was added to thecuvette and the media plus cells were transferred to a 48 wellnon-tissue culture treated plates and then incubated at 37° C. Geneediting was measured by high-throughput DNA sequencing of PCR ampliconsof the HBB gene. Percent gene modification by non-homologous end joining(“NHEJ”, caused by the healing of a double stranded break in the DNAfollowing ZFN-induced cleavage) or targeted integration of the oligofollowing ZFN cleavage (“gene correction”) was analyzed.

As shown in FIG. 4, the β globin oligo donor was inserted into theproper locus, as verified by the presence of the novel restriction sitepresent on the donor DNA. Furthermore, sequence analysis was performedto verify the percent of alleles comprising a targeted integrationand/or indels (insertions and/or deletions) introduced by error-proneNHEJ following ZFN cleavage. Genomic DNA was extracted (using QiagenQIAamp® DNA micro kit) and analyzed for ZFN activity as follows.Briefly, the region comprising the cleavage site was amplified by PCR bystandard methods, and following amplification, the PCR product wassequenced via MiSeq high throughput sequencing analysis according tomanufacturer's instructions (Illumina®).

To quantitate the percent of edited alleles, the genomic region ofinterest was PCR amplified using primers which add the standardIllumina® sequencing adapter sequences. A second group of 13 rounds ofPCR was performed to add barcode and bridge adapter sequences to bothends. Sequencing was performed on an Illumina® MiSeq according tomanufacturer's protocols for amplicon sequencing. The MiSeq generatespaired-end reads, which are merged and adapter-trimmed using a standardalignment software. Reads were then demultiplexed by sample via barcodesequence pairs using custom scripts. Amplicon sequences were thenglobally aligned to a reference sequence via an implementation of theNeedleman-Wunsch algorithm (Needleman, Saul B.; and Wunsch, Christian D.(1970). Jour Mol Bio 48 (3): 443-53). Gaps or insertions in thealignment were counted as % NHEJ events, and compared to an untreatedcontrol sample sequence to determine sequence-specific background rates.

For calculation of targeted integration, Amplicon sequences wereglobally aligned to a reference sequence via a biopython implementationof the Needleman-Wunsch algorithm (Needleman, Saul B.; and Wunsch,Christian D., ibid). Sequence changes generated via experimentaltreatments were searched for, counted, and compared to counts in controlsamples. Known single feature polymorphisms (SNPs) may be masked outduring this process and excluded from further counts (e.g., 1-bpdeletion SFPs close to the ZFN target site). The percent ofnon-homologous end joining (NHEJ %) (also referred to as indels) wascalculated by determining the percentage of sequences that containinsertions and/or deletions. Samples treated only with GFP vector wereused to assess the PCR and sequencing error based background frequencyof insertions and deletions. Background frequencies of less than 1% wereobserved.

The results demonstrate that in the presence of the oligo donor and theZFN pair, approximately 12-14% of the cells showed an integration event.In addition, analysis for the presence of indels showed that when ZFNswere present, indels were detected in 42-70% of the alleles. See, also,FIG. 4.

To differentiate the transgenic CD34+ cells into mature RBCs, methodsknown in the art were used. Pools of ZFN-modified CD34+ cells wereinduced to differentiate as described in Giarratana et al. (2011) Blood118(19):5071-9.

At day 14 of erythrocyte differentiation, total RNA was isolated andsplicing of the beta globin gene was analyzed via standard RT-PCR andsequence analysis as described above. The results (see Table 2)demonstrate that cleavage by the ZFNs alone is capable of introductionof indels that interfere with proper splicing, and this effect is alsoseen in the presence of the IVS 1-1 oligo donor, where a percent ofsplicing occurs via novel cryptic splicing sites (see FIG. 5). However,when the WT oligo donor is used, splicing is partially restored to thelevels seen when ZFNs are not used. In addition, the presence of theintroduced XbaI site in the oligo donor does not appear to affectsplicing.

TABLE 2 Splicing products in treated erythrocytes Percent of splicingproducts Sample Normal Cryptic 1 Cryptic 2 Other ZFN only 86.6 9.3 2.51.6 ZFN + Oligo_G-A 90.2 6.5 1.7 1.6 ZFN + Oligo_G-A Xbal 91.8 5.3 1.81.2 ZFN + Oligo_WT Xbal 94.4 3.9 0.9 0.8 Oligo_G-A 99.6 0.1 0.1 0.2Oligo_G-A Xbal 99.7 0.1 0.1 0.1 Oligo_WT Xbal 99.7 0.1 0.1 0.2 GFP 99.70.1 0.1 0.2 Untransfected 99.3 0.3 0.2 0.3

Example 4 Oligo Based Gene Correction of the IVS1-1 Mutation inThalassemia CD34+ Cells

Thalassemia patient derived CD34+ cells were then treated to correct theIVS 1-1 mutation. This patient, “p13”, is a β⁰β⁰ patient with the IVS1-1mutation on one allele and a nonsense mutation (TGG->TAG) on the otherallele at codon 15. CD34+ cells were isolated and treated with thenucleases and donor oligos as described in Example 3. In addition, highthroughput sequencing analysis was performed to look at the modificationcaused by the nucleases and oligos. Genomic DNA was isolated from thesamples and demonstrated that there was a high rate of cleavage with theXbaI restriction enzyme due to targeted integration of the oligo donor(see FIG. 6).

Sequence analysis is shown below in Table 3. The first set of data isfor the WT CD34 cells where both beta globin alleles have wild typesequence, whereas the second data group is for the beta thalassemia p13patient cells.

TABLE 3 Results following gene correction in CD34+ cells Cell % “G” atType ZFNs Oligo % Indel Xbal RFLP IVS11 site WT 43545 + none 58.4 0.579.2 C34 43544 43545 + none 18.1 0.3 94.2 45946 43545 + none 4.3 0.098.6 45952 43545 + WT-Xbal 37.5 9.7 91.0 43544 43545 + WT-Xbal 5.5 1.498.7 45946 43545 + WT-Xbal 1.0 0.1 99.8 45952 GFP WT-Xbal 0.6 0.0 99.8Untransfected none 0.1 0.0 99.9 % % Indel % % Indel “G” at at Xbal at %Xbal- IVS11 IVS11 RFLP IVS11 RFLP % Xbal site on site on on site on onIndel RFLP IVS1.1 IVS1.1 IVS1.1 codon15 codon15 ZFNs Oligo overalloverall allele allele allele allele allele IVS1.1 43545 + none 73.7 1.210.6 85.0 1.9 60.6 0.6 P13 43544 43545 + none 39.8 0.8 11.6 67.2 1.422.4 0.5 45946 43545 + none 18.6 0.5 10.5 48.2 1.5 5.0 0.1 45952 43545 +WT- 55.9 21.7 34.6 82.7 29.5 37.6 16.6 43544 Xbal 43545 + WT- 12.6 4.018.0 33.8 10.9 5.3 1.6 45946 Xbal 43545 + WT- 3.2 0.9 13.9 11.6 3.4 0.90.3 45952 Xbal GFP WT- 0.1 0.0 8.9 0.0 0.0 0.2 0.0 Xbal 43545 + WT 19.10.0 33.5 39.3 0.1 8.3 0.0 43544 un- none 0.1 0.0 10.7 0.1 0.0 0.2 0.0transfected

The data demonstrates activity of the nucleases at the IVS1-1 site inboth cell populations, as measured by indel activity in the ZFN alonesamples. In the presence of the donors comprising the XbaI site, thereis a notable amount of RFLP detection, indicating integration of thedonor oligo. In the p13 cells, use of the WT oligo donor that includesthe XbaI site causes the appearance of the XbaI RFLP on both alleles.There is also an increase in WT sequence “G” at the IVS1-1 site on theIVS1-1 allele (for example, with the 43545/43544 ZFN pair, a value of34.6% WT sequence was detected as compared to 10.7% in the untransfectedcells.

Patient-derived CD34+ cells that have been ZFN-modified were induced todifferentiate into colonies using Stemcell Technologies' Methocultmethylcellulose medium according to manufacturer's directions.Differentiation was analyzed by assay of colony types arising fromMethocult-induced differentiation: colony-forming units, erythroid(“CFU-E”); burst-forming units, erythroid (“BFU-E”); colony-formingunits, granulocyte/macrophage (“CFU-GM”) and colony-forming units;granulocyte/erythrocyte/monocyte/macrophage (“CFU-GEMM”). The BFU-Ecolonies were picked and analyzed for genotyping and beta-globinsplicing using MiSeq analysis. 20% of all the clones have IVS1.1mutation corrected to wild type “G”, and the beta-globin splicingrestored to normal. The data also confirmed that addition of the XbaIrestriction site in the IVS1 intron has no adverse effect on beta-globinsplicing.

Taken together, these data demonstrate successful gene correction of amutation (the IVS1-1 mutation) in beta globin gene in beta thalassemiapatient-derived CD34+ cells.

All patents, patent applications and publications mentioned herein arehereby incorporated by reference in their entirety.

Although disclosure has been provided in some detail by way ofillustration and example for the purposes of clarity of understanding,it will be apparent to those skilled in the art that various changes andmodifications can be practiced without departing from the spirit orscope of the disclosure. Accordingly, the foregoing descriptions andexamples should not be construed as limiting.

What is claimed is:
 1. A genetically modified cell comprising a genomicmodification, wherein the genomic modification corrects at least onemutation in an endogenous aberrant human beta-hemoglobin (Hbb) gene andfurther wherein the genomic modification is selected from the groupconsisting of insertions, deletions and combinations thereof.
 2. Thegenetically modified cell of claim 1, wherein the mutation is in anintervening sequence 1, mutation number 1 (IVS1-1) mutation, an IVS1-5mutation, an IVS1-6 mutation, an IVS1-110 mutation, an IVS2-1 mutation,an IVS2-745 mutation or an IVS2-654 mutation.
 3. The geneticallymodified cell of claim 2, wherein the genomic modification corrects apoint mutation.
 4. The genetically modified cell of claim 3, wherein thegenomic modification is within one or more of the sequences shown in SEQID NO:3.
 5. The genetically modified cell of claim 4, wherein thegenomic modification within SEQ ID NO: 3 disrupts a splice donor motif.6. The genetically modified cell of claim 5, wherein the GT dinucleotideat the beginning of intron 1 of the human beta-globin gene is changed toan AT dinucleotide.
 7. The genetically modified cell of claim 3, whereinthe genomic modification corrects a mutation that alters the splicedonor site for mRNA processing of the aberrant Hbb gene that leads tobeta-thalassemia.
 8. The genetically modified cell of claim 3, whereinthe genomic modification is at or near any of the sequences shown as SEQID Nos. 4 or
 5. 9. The genetically modified cell of claim 8, wherein thegenomic modification comprises insertion of donor sequence comprising anovel restriction enzyme cleavage site with respect to the endogenousHbb gene.
 10. The genetically modified cell of claim 9, wherein thedonor sequence is between 2 kb to 200 kb in length.
 11. The geneticallymodified cell of claim 10, wherein the donor sequence is selected fromSEQ ID Nos. 20 or 22 and the novel restriction enzyme cleavage site isan Xba I site.
 12. The genetically modified cell of claim 1, wherein thecell is a stem cell.
 13. The genetically modified cell of claim 12,wherein the stem cell is a hematopoietic stem cell.
 14. The geneticallymodified cell of claim 13, wherein the hematopoietic stem cell is aCD34+ cell.
 15. A genetically modified differentiated cell descendedfrom the stem cell of claim
 12. 16. The genetically modified cell ofclaim 15, wherein the cell is a red blood cell (RBC).
 17. Thegenetically modified cell of claim 1, wherein the genomic modificationis made by a nuclease.
 18. The genetically modified cell of claim 17,wherein the nuclease comprises at least one zinc finger nuclease (ZFN),TALEN or CRISPR/Cas system.
 19. The genetically modified cell of claim17, wherein the nuclease is introduced into the cell as apolynucleotide.
 20. The genetically modified cell of claim 1, whereinthe insertion comprises integration of a donor polynucleotide encoding atransgene.
 21. The genetically modified cell of claim 17, wherein thenuclease comprises a zinc finger nuclease, the zinc finger nucleasecomprising 4, 5, or 6 zinc finger domains, each zinc finger domaincomprising a recognition helix region and further wherein therecognition helix regions are in the order shown in one of the last tworows of Table
 1. 22. A pharmaceutical composition comprising thegenetically modified cell of claim
 17. 23. A zinc finger proteincomprising 4, 5, or 6 zinc finger domains comprising a recognition helixregion, wherein the zinc finger proteins comprise the recognition helixregions in the order shown in the last two rows of Table
 1. 24. A fusionprotein comprising a zinc finger protein of claim 23 and a wild-type orengineered cleavage half-domain.
 25. A polynucleotide encoding one ormore proteins of claim
 23. 26. An isolated cell comprising one or morefusion proteins of claim 24 or polynucleotides encoding said fusionproteins.
 27. The cell of claim 26, wherein the cell is selected fromthe group consisting of a red blood cell (RBC) or a precursor cell. 28.A kit comprising at least one a polynucleotide according to claim 25.29. A method of altering Hbb expression in a cell, the methodcomprising: introducing, into the cell, one or more polynucleotidesaccording to claim 25, under conditions such that the one or moreproteins are expressed and expression of the Hbb gene is altered. 30.The method of claim 29, wherein expression of the Hbb gene is increased.31. The method of claim 29, further comprising integrating a donorsequence into the genome of the cell.
 32. The method of claim 31,wherein the donor sequence is introduced to the cell using a viralvector, as an oligonucleotide or on a plasmid.
 33. The method of claim29, wherein the cell is selected from the group consisting of a redblood cell (RBC) precursor cell and a hematopoietic stem cell.
 34. Themethod of claim 31, wherein the donor sequence comprises a transgeneunder the control of an endogenous or exogenous promoter.
 35. The methodof claim 31, wherein the donor sequence comprises an oligonucleotidethat corrects a mutation that alters the encoded splice donor site inthe Hbb gene used for mRNA processing.
 36. A method of producing agenetically modified cell comprising a genomic modification within anendogenous Hbb gene, the method comprising the steps of: a) contacting acell with a polynucleotide according to claim 25, b) subjecting the cellto conditions conducive to expressing the fusion protein from thepolynucleotide; and c) modifying the endogenous Hbb gene with theexpressed fusion protein sufficient to produce the genetically modifiedcell.
 37. The method of claim 36, wherein the method further comprisesstimulating the cells with at least one cytokine.
 38. The method ofclaim 36, wherein the method further comprises the step of deliveringthe polynucleotide inside the cell.
 39. The method of claim 38, whereinthe delivery step comprises use of at least one of a non-viral deliverysystem, a viral delivery system, and a delivery vehicle.
 40. The methodof claim 36, wherein the delivery step further comprises subjecting thecells to an electric field.
 41. A kit for performing the method of claim36, the kit comprising: at least one polynucleotide encoding a fusionprotein comprising a zinc finger nuclease comprising 4, 5, or 6 zincfinger domains in which each of the zinc finger domains comprises arecognition helix region in the order shown in the last two rows ofTable 1, and optionally, directions for using the kit.
 42. A method oftreating a patient with β-thalassemia, the method comprisingadministering to the patient the pharmaceutical preparation of claim 22in an amount sufficient to increase the Hbb gene expression in thepatient.